WO1994023029A1 - Sequence de nucleotides codant une proteine chromosomique, proteines chromosomiques associees et leurs utilisations - Google Patents

Sequence de nucleotides codant une proteine chromosomique, proteines chromosomiques associees et leurs utilisations Download PDF

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WO1994023029A1
WO1994023029A1 PCT/AU1994/000158 AU9400158W WO9423029A1 WO 1994023029 A1 WO1994023029 A1 WO 1994023029A1 AU 9400158 W AU9400158 W AU 9400158W WO 9423029 A1 WO9423029 A1 WO 9423029A1
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mitosis
protein
antigen
nucleic acid
cells
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PCT/AU1994/000158
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English (en)
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Jing Ping Yeo
Frank Alderuccio
Ban-Hock Toh
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Monash University
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Priority to AU63718/94A priority Critical patent/AU6371894A/en
Publication of WO1994023029A1 publication Critical patent/WO1994023029A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • a NUCLEOTIDE SEQUENCE ENCODING A CHROMOSOMAL PROTEIN, ASSOCIATED CHROMOSOMAL PROTEINS AND THEIR USES
  • the present invention relates to a novel isolated nucleic acid molecule encoding a mitosis-associated nuclear antigen and anti-sense sequences thereof, to mitosis associated proteins and components thereof.
  • the invention also extends to methods of disrupting mitosis using the anti-sense sequences and molecules immunologically reactive to said proteins.
  • Mitosis is a sequence of events in a cell which results in the division of the cell nucleus and is responsible for asexual cell division. During mitosis the nuclear DNA condenses into chromosomes which attach to spindles and are pulled apart, ultimately allowing two nuclei to form.
  • mitotic spindle requires conversion of the relatively stable microtubule arrays in interphase cells into the more dynamic, transient microtubular fibres of the spindle 1-2 . Chromosomal segregation is thought to be mediated by microtubule "motor" proteins which attach to the microtubule fibres 3 . While much has been learnt about the mechanochemical properties of the spindle, the regulation of its assembly has remained largely unknown 4 - 5 . A regulatory role for cdc2 kinase in mitotic spindle assembly has been suggested 6 .
  • chromosomes in spindle assembly An essential requirement for chromosomes in spindle assembly is suggested by the reduction in the number of kinetochore as well as non- kinetochore microtubules following removal of individual chromosomes from insect spermatocytes by micromanipulation, a reduction which is quantitatively dependent upon the number of chromosomes removed 10 .
  • the inventors cloned a cDNA encoding a novel, and evolutionarily conserved, chromosomal protein which is necessary for mitotic spindle assembly.
  • the protein is about 47 kD, a physiological substrate for cdc2 kinase, is phosphorylated only during mitosis and is herein referred to as the mitosis-associated nuclear antigen.
  • the inventors also identified polypeptide components with a molecular weight of 31 kD, 67 kD and 200 kD which immunoprecipitate together.
  • the 31 kD polypeptide has phosphatase activity whereas the 67 and 200 kD polypeptides are phosphorylated with the mitosis- associated nuclear antigen. Furthermore it has been discovered that the 31 kD polypeptide is activated by the action of cdc2 kinase through the phosphorylation of the 47 kD polypeptide. These observations suggest that, collectively, the 47 kD, 31 kD, 67 kD and 200 kD polypeptides constitute a functional multicomplex.
  • one aspect of the present invention is directed to a novel isolated nucleic acid molecule encoding a mitosis-associated nuclear antigen.
  • nucleic acid molecule of the present invention encodes a mitosis-associated antigen of about 47 kD, which antigen is phosphorylated in mitosis and is a substrate for cdc2 kinase.
  • nucleic acid molecule of the invention encodes an open reading frame of about 1254 bp. More preferably said antigen encoded comprises at least three S/T-P sequences, ten consecutive lysine residues, the amino acid sequence KKKQRK and has a neutral PI.
  • the invention also relates to an isolated nucleic acid molecule complementary to the molecule encoding the mitosis- associated nuclear antigen and to an isolated nucleic acid molecule hybrid!sable therewith.
  • the invention relates to the isolated nucleic acid molecule wherein the nucleic acid sequence is that represented by Figure 4 or a part thereof.
  • the invention provides an isolated protein comprising a mitosis-associated nuclear antigen of about 47 kD which is capable of being phosphorylated during mitosis and is capable of being a substrate for cdc2 kinase.
  • the present invention relates to an isolated protein associated with a mitosis-associated nuclear antigen wherein said protein is about 31 kD and demonstrates phosphatase activity.
  • the present invention relates to an isolated protein associated with a mitosis-associated nuclear antigen wherein said protein is about 67 kD and is capable of being phosphorylated during mitosis.
  • the present invention relates to an isolated protein associated with a mitosis-associated nuclear antigen wherein said protein is about 200 kD and is capable of being phosphorylated during mitosis.
  • the invention provides immunologically reactive molecules reactive with the proteins of the invention.
  • the invention relates to a method of disrupting or arresting mitosis in cells comprising administering an effective amount of a mitosis-associated nuclear antigen blocking agent to said cells wherein said mitosis associated nuclear antigen is about 47 kD and is capable of being a substrate for cdc2 kinase and wherein said blocking agent is a molecule capable of preventing said antigen from performing its role in mitosis.
  • the invention in a yet further aspect relates to a method of disrupting or arresting mitosis in cells comprising administering an effective amount of an associated protein blocking agent to said cells, wherein said associated protein is of about 31 kD and is capable of phosphatase activity or is about, 67 or 200 kD and is capable of being phosphorylated during mitosis and wherein said associated protein is capable of association with a mitosis-associated nuclear antigen of about 47 kD which antigen is a substrate for cdc2 kinase, and wherein said blocking agent is a molecule capable of preventing said associated protein from performing its role in mitosis.
  • an associated protein blocking agent is a molecule capable of preventing said associated protein from performing its role in mitosis.
  • the invention also extends to a method of treatment of cancer cells in a patient comprising administering an effective amount of a mitosis-associated nuclear antigen blocking agent or an associated protein blocking agent to said patient such that mitosis is arrested.
  • the invention provides a pharmaceutical preparation for use in the methods.
  • the invention provides a method of identifying or localising dividing cells, particularly rapidly dividing cells, in a sample which method comprises adding an effective amount of a mitosis-associated nuclear antigen binding agent, wherein said agent is specific for the antigen in its mitosis associated state, and detecting presence of binding of said antigen.
  • the invention provides a method of detecting autoimmune disease in a subject where an autoantibody directed to a protein of about 47 kD which protein is a substrate of cdc2 kinase is indicative of the presence of said disease, said method including contacting a sample from the subject likely to contain the autoantibody with an isolated preparation of the protein for a time and under conditions sufficient for a complex to form and detecting presence of said complex.
  • the invention provides a method of diagnosing autoimmune disease in a patient where an autoantibody reactive with a protein of about 47 kD which is a substrate for cdc2 kinase is correlated with the presence of said disease, said method including contacting a sample from said patient with an isolated preparation of the protein for a time and under conditions sufficient for a complex to form and detecting said complex.
  • the autoimmune disease may be any disease wherein autoantibody is implicated in the disease process and may include diseases such as systemic lupus erythematosus, rheumatoid arthritis, schleroderma, polymyositis, dermatomyositis and mixed connective tissue disease.
  • the invention provides a kit for detecting autoimmune disease in a patient wherein an autoantibody reactive with a protein of about 47 kD which protein is a substrate for cdc2 kinase, is correlated with said disease, comprising a first compartment adapted to act as a solid support for an isolated preparation of the protein and is adapted to receive a sample from said patient suspected of containing said autoantibody, and at least one other compartment adapted to contain appropriate ⁇ reagents and buffers for detecting a complex which may be formed by said autoantibody and said protein.
  • the invention provides a kit for analysis of mitosis or karyotype analysis containing a mitosis-associated nuclear antigen blocking agent and a detector means.
  • Fig. 1 shows immunofluorescence localisation of RMSA-1 auto antigen to chromosomes of mitotic cells.
  • Human Hep 2 cells Kerallestad
  • autoimmune serum from a patient with discoid lupus erythematous (diluted 1:200 in phosphate-buffered saline, PBS), and traced with fluorescein- isothiocyanate-labelled anti-human immunoglobulin (Wellcome).
  • the cells were washed and counterstained with 5 ⁇ M Hoechst 33342 DNA dye. Bar: 2 ⁇ m
  • Fig. 2 shows the biochemical characterisation of RMSA-1.
  • B) Immunoblot reactivity with the 47 kD antigen in HeLa cells (lane 1) is confined to the nuclear (lane 2) and crude and purified karyoplast fractions (lanes 4 and 6 respectively) but not in the crude cytoplasmic fraction (lane 3) nor in the non-karyoplast cytoplasmic fraction (lanes 5 and 7).
  • the autoimmune serum immunoprecipitates the 47 kD antigen and associated molecules of 31 kD, 67 kD and 200 kD from extracts of L-[ 35 S]-methionine/cysteine-labelled interphase HeLa cells (I, lane 3) and metaphase arrested. (M, lane 4) cells; lanes 1, 2 normal human IgG immunoprecipitates. The same molecules were immunoprecipitated from extracts of 32 P-orthophosphate-labelled, metaphase-arrested, HeLa cells (M, lane 6) but not from interphase cells (I, lane 5); normal human serum controls (lanes 7 and 6).
  • the autoimmune serum (lane 2, 4) immunoblots a 31K fusion protein generated by pJPL4 (lane 2) and pJPL41.6 (lane 4); lanes 1, 3, normal human serum controls. Autoantibody, affinity-purified from ⁇ JPL4 fusion protein, reacts with a 47K antigen in HeLa cells (lane 6); material eluted from a non-immunoreactive clone is unreactive (lane 5).
  • Fig. 3 Mitotic arrest and prevention of mitotic spindle formation in mouse L cells induced by antisense mRNA or by autoantibody.
  • A Progressive rounding and detachment of antisense-transfected cells (b-e) and of cells electroporated with affinity-purified autoantibody (g-j).
  • Antisense- transfected L cells were treated with ZnCl 2 for 3 (b), 6 (c), 12 (d) and 24 h (e).
  • a Sense-transfe ⁇ ted cells treated with ZnCl 2 for 24 h.
  • f Cells electroporated with normal human IgG and examined at 24 h. Phase-contrast micrographs. Scale bar, 5 ⁇ m.
  • B Mitotic arrest of L cells 24 h after induction of antisense mRNA by ZnCl 2 (a) or electroporation with affinity- purified autoantibody (b). Floating cells were collected, cytocentrifuged (700g for 5 min) and stained with 5 ⁇ M Hoechst DNA dye. Scale bar, 2 ⁇ m.
  • C Immunoblot of antisense transfected cells (AS) at 3, 6, 12 and 24 h after treatment with ZnCl 2 showing absence of RMSA-1 at 12 h; S, immunoblot of sense transfected cells treated with ZnCl 2 ; D, Flow cytometry showing cell-cycle arrest at G2/M.
  • Sense- and antisense- transfected L cells were synchronized by double thymidine block and treated with ZnCl 2 .
  • L cells were electroporated with affinity-purified autoantibody (DLE) or normal human IgG (NHS). Cells were sampled from 3 to 24 h. Only the 24-h time point is shown.
  • Fig. 4 Molecular characterization of RMSA-1.
  • a Relationship of pJPL41.6 and pJPZl to pJPZlL4, the 'full length' cDNA clone (accession number, L26953) encoding the RMSA-1 polypeptide. Restriction sites are Aval (A), EcoRI (E), Hindlll (H), Pstl (P) and X ⁇ oI (X).
  • b Northern blot analysis of total HeLa cell RNA with 32 P-labelled cDNA insert of pJPZlL4 (lane 1) reveals a single mRNA species of about 3 kb; control blot using 32 P-labelled actin probe (lane 2).
  • Positions of 18S and 28S rRNA are indicated.
  • c Complete nucleotide and deduced amino-acid sequence of RMSA-1. Underlined are the first inframe stop (TGA) codon, potential nuclear localization signals KKKKKKKKKK and KKKQRK an putative polyadenylation signal AATAAA.
  • TGA first inframe stop
  • KKKKKKKKKKKK and KKKQRK potential nuclear localization signals
  • AATAAA putative polyadenylation signal
  • Three consensus S/T-P motifs for phosphorylation by p34 cdc2 kinase are boxed.
  • Fig. 5 Identification of RMSA-1 complex as a type I phosphatase by 35 S labelling, immuno-precipitation and immunoblot procedures of nuclear extracts of interphase cells (marked I) and mitotic cells (marked M).
  • FIG. 6 Graphic representations of phosphatase activity of RMSA-1 complex.
  • Fig. 7 Autoradiographs of RMSA-1 immunoprecipitates of nuclear phosphoproteins and graphic representations of phosphatase activity.
  • the present invention is directed to an isolated nucleic acid molecule encoding a mitosis associated nuclear antigen, wherein said antigen is about 47 kD, is capable of being phosphorylated during mitosis and capable of being a substrate for cdc2 kinase.
  • the antigen appears only to be phosphorylated during mitosis.
  • isolated means at least some purification away from the other nucleic acids and/or proteins.
  • the nucleic acid molecule has an open reading frame of about 1254 bp. More preferably the antigen encoded comprises at least three S/T-P sequences, ten consecutive lysine residues, the amino acid sequence KKKQRK and has neutral PI. Still more preferably the antigen encoded comprises about 418 amino acid residues.
  • the antigen, or part thereof encoded by the nucleic acid molecule of the invention may be in the form of a fusion protein.
  • This aspect of the invention also relates to nucleotide sequences complementary to the nucleic acid molecule encoding the mitosis-associated nuclear antigen, derivatives, variants, homologues, analogues and fragments thereof which are functionally related to said nucleic acid molecule.
  • the term "functionally related" means that the nucleic acid derivative, variants, homologue, analogue or fragment has a biological function comparable to that of the nucleic acid encoding the mitosis-associated nuclear antigen or encodes a peptide with a biological function comparable to that of the mitosis- associated nuclear antigen.
  • nucleic acid molecules which differ from the nucleic acid molecule encoding the 47 kD mitosis-associated nuclear antigen herein described in the nucleotides and amino acids encoded thereby but are functionally related thereto.
  • nucleotides used herein extends to modified nucleic acids with a sulphur backbone (phosphorothioates) and with methyl groups (methylphosphorothioates). This aspect of the present invention also extends to nucleic acid sequences capable of hybridising under low, preferably under medium and most preferably under high stringency conditions to the nucleic acid molecule of the invention.
  • the level of stringency is generally described by Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y. USA.
  • a low stringency is defined herein as being 2 x SSC, 0.1-0.5% w/v SDS at 37-45 ⁇ C for 30 to 60 mins.
  • Medium stringency is considered herein to be 0.5 x SSC, 0.25%-0.5% w/v SDS at > 45"C for 30 to 60 mins and high stringency 0.1 x SSC, 0.25-0.5% w/v SDS at > 45°C for 30 to 60 mins.
  • the nucleic acid molecule of the invention may be in the form of DNA or RNA, in single stranded or double stranded form, linear or covalently closed circular form.
  • the nucleic acid molecule of the invention may be carried on a vector including an expression vector, such as a plasmid, viral vector such as a retrovirus, cosmid or other convenient vector.
  • an expression vector such as a plasmid, viral vector such as a retrovirus, cosmid or other convenient vector.
  • the invention also extends to a host cell transformed with the vector described.
  • the invention relates to a novel isolated nucleic acid molecule of the sequence shown in Fig. 4, and to derivatives, variants, homologues, analogues and fragments thereof.
  • the product encoded by this preferred aspect of the invention shown in Fig. 4 is designated RMSA-1 (Regulator of Mitotic Spindal Assembly-1).
  • the present invention relates to a nucleic acid molecule encoding RMSA-1 or the anti-sense, or partial anti-sense sequence of RMSA-1 comprising a vector.
  • the vector may be a viral vector which is capable of reproduction in human cells, a plasmid or any other delivery agent which is capable of delivering RMSA-1 into cells such as a growth factor which binding to its receptor facilitates entry into the target cells.
  • the invention relates to pJPZlL4, pJPL41.6, pJPZl, pJPZ2 and ⁇ JPL4 as described herein.
  • nucleic acid sequence of the nucleic acid molecules of the invention may be the same as naturally occurring sequence for RMSA-1 or may contain single or multiple nucleotide substitutions, deletions and/or additions thereto.
  • the nucleotide derivatives contemplated may encode insertional derivatives of RMSA-1 variant polypeptides or proteins and such derivatives include amino acid and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterised by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place and these are well known by a skilled person. Typical substitutions are those made in accordance with Table 1 below. TABLE 1 Suitable residues for amino acid substitutions
  • the invention relates to an anti-sense sequence of the nucleic acid molecule encoding the mitosis-associated antigen of the invention.
  • the anti-sense sequence is in the form of RNA or DNA such that it is capable of blocking transcription or translation of the mitosis-associated nuclear antigen gene.
  • the anti-sense sequence selectively binds the m-RNA of the mitosis-associated antigen, or a part thereof, and prevents translation of the m-RNA into protein.
  • the anti-sense sequence is capable of associating directly with the gene encoding the mitosis- associated antigen, or a part thereof, in order to block transcription of the gene, such as for example, by triplex formation between the oligonucleotide anti-sense sequence and double stranded DNA.
  • the anti-sense sequence has a ribozyme capacity such that the gene encoding the mitosis-associated nuclear antigen is inactivated.
  • the anti-sense sequence can be delivered by a delivery vehicle or carrier such as a plasmid or a viral vector capable of reproduction in human cells, such as a retrovirus, or any other agent which is capable of delivering the sequence into target cells including liposomes.
  • the anti-sense sequence may be carried on an inducible vector such as pMT 4 SV neo or other inducible vectors so that timing of the production of the anti-sense sequence can be controlled. More preferably the anti-sense sequence spans the promoter and/or start sequence of the nucleic acid molecule encoding the mitosis-associated antigen.
  • the anti-sense sequence may be the full length sequence, partial sequence or of any convenient size and is preferably in the region of 10-20 bp. Preferably the anti-sense sequence spans the promoter and/or start sequence of the RMSA-1 gene shown in Fig. 4.
  • the antisense oligonucleotide comprises the antisense sequence around the transcription start codon of Fig. 4. More preferably the oligonucleotide comprises the following sequence ATTCCCCCATCAGGATT or AUUCCCCCAUCAGGAUU.
  • the anti-sense sequence may be modified, such as chemically, in order to allow improved delivery efficiency in a therapeutic context. Such modifications as those known to a skilled addressee are included within the scope of this invention and may include using sulphur instead of oxygen in the phosphodiester backbone of the nucleic acid ( phosphorothioates ) or methyl groups (methylphosphorothioates).
  • the anti-sense sequence of the invention may be used in arresting mitosis, in diagnostic applications or cancer therapy and in karyotype analysis as discussed below.
  • the nucleic acid molecule of the invention may be labelled with a reporter molecule providing, under suitable conditions, a detectable signal.
  • reporter molecules include radio nucleotides, chemiluminescent molecules, bioluminescent molecules, fluorescent molecules or enzymes. Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase amongst others.
  • the isolated nucleic acid molecule comprises at least one of a primer pair, said pair being capable, under suitable conditions, of facilitating replication of a gene encoding a mitosis-associated nuclear antigen, wherein said antigen is about 47 kD and is capable of being a substrate for cdc2 kinase.
  • the present invention also extends to polypeptides, proteins, fragments thereof antigenic regions and haptens encoded by the nucleic acid molecule of the invention.
  • the invention provides an isolated mitosis-associated nuclear antigen of about 47kD which is capable of being phosphorylated during mitosis and is capable of being a substrate for cdc2 kinase.
  • the antigen comprises at least 3 S/T-P sequences, ten consecutive lysine residues, the amino acid sequence KKKQRK and has a neutral PI.
  • the antigen comprises about 418 amino acid residues.
  • the isolate is biologically pure which means a preparation of the antigen comprising at least 20%, preferably at least 30%, more preferably at least 40%, even more preferably at least 60%, still more preferably greater than 80% relative to other components as determined by weight, mitosis-association activity, antibody reactivity or other convenient means.
  • the invention comprises a mitosis-associated nuclear antigen or derivatives, variants, homologues and analogues including all naturally occurring and artificially created protein molecules, fragments, antigenic determinants and haptens thereof with the following sequence:
  • derivatives, variants, homologues, analogues and fragments thereof have the same meaning, when applied to proteins, as given above.
  • the antigen of the invention is present in what appears to be a complex or multicomplex with three other protein molecules of about 31, 67 and 200 kD. These associated proteins are present in cell nuclear extracts and appear to be involved in spindle formation. These other protein molecules appear to be specifically associated with the antigen of the invention and are referred to herein as "associated proteins".
  • the present invention also relates to an isolated preparation of one or more of the associated proteins as herein described.
  • isolated has the same meaning as described above.
  • the present invention relates to an isolated protein capable of association with a mitosis associated nuclear antigen of about 47 kD, which antigen is capable of being a substrate for cdc2 kinase, wherein said protein is about 31 kD and is capable of phosphatase activity.
  • said 31 kD protein is a type 1 phosphatase activated by phosphorylation of the 47 kD protein by cdc2 kinase during mitosis.
  • said 31 kD protein is a RMSA-1 associated phosphatase as herein described.
  • the present invention relates to an isolated protein capable of association with a mitosis- associated nuclear antigen of about 47 kD, which antigen is capable of being a substrate for cdc2 kinase, wherein said protein is about 67 kD and is capable of being phosphorylated during mitosis.
  • the present invention relates to an isolated protein associated with a mitosis capable of association nuclear antigen of about 47 kD, which antigen is capable of being a substrate for cdc2 kinase, wherein said protein is about 200 kD and is capable of being phosphorylated during mitosis.
  • the present invention relates to an isolated complex of proteins said complex comprising the following proteins: a mitosis-associated nuclear antigen of about 47 kD, a protein of about 31 kD which is capable of phosphatase activity, a protein of about 67 kD and a protein of about 200 kD wherein the proteins of about 67 kD and 200 kD are capable of being phosphorylated during mitosis.
  • the present invention also extends to immunologically reactive molecules (IRMs) which are immunologically reactive to the mitosis-associated nuclear antigen of the invention or to the associated proteins as herein described.
  • IRMs immunologically reactive molecules
  • an IRM specific for the antigen of the invention or one of the associated proteins as herein described, or fragments, antigenic determinants or haptens thereof.
  • the IRM of the invention may be labelled with a reporter molecule, providing under suitable conditions, a detectable signal as described above.
  • the IRM of the invention may be associated with a toxic agent.
  • associated in this context means that the toxic agent is in combination with, coupled to, linked to, or bonded with the IRM.
  • the toxic agent is any agent which is noxious to cells and includes radionuclides, cytotoxins, cytostatic agents, cytoinhibiting agents and cytomodulating agents.
  • the invention comprises an IRM specific for one or more antigenic determinants or haptens which are unique to the mitosis associated antigen, or one of the associated proteins of the invention.
  • said IRMs are antibodies, more preferably monoclonal antibodies (MABs), directed towards one or more antigenic determinants or haptens of said mitosis associated antigen or one of said associated proteins.
  • said IRMs are useful in detecting and arresting mitosis and therefore have applications in diagnosis of tissues containing rapidly dividing cells and in cancer therapy and karyotype analysis.
  • Antibodies and other IR 's of the present invention may be of any animal origin including mammal- such as humans, live stock animals, companion animals, wild animals and laboratory test animals (eg. mice, rats, rabbits and guinea pigs).
  • mammal- such as humans, live stock animals, companion animals, wild animals and laboratory test animals (eg. mice, rats, rabbits and guinea pigs).
  • An "animal” also extends to non-mammalian species such as birds (eg. chickens and other poultry, emus and ostriches).
  • antibody includes naturally occurring antibodies, recombinant antibodies, synthetic antibodies including fusions or chimers of antibodies and fragments of any of the foregoing such as Fab and F(ab' ) 2 and chimers of these optionally together with any other molecule or agent which can facilitate delivery of the IRM into the cell.
  • the antibody IRM is a recombinant form
  • the molecule may be encoded by a naturally occurring or synthetic nucleotide sequence and expressed in any convenient expression system.
  • the molecule is synthetic, it is conveniently prepared by the step-wise addition of single amino acid groups or amino acid fragments of, for example antibodies. With regard to the latter, the synthetic antibody may be a fusion or chimeric antibody comprising light or heavy chains derived from other antibodies.
  • the present invention also extends to a method of disrupting or arresting mitosis.
  • the invention provides a method of disrupting or arresting mitosis in cells comprising administering an effective amount of a mitosis-associated nuclear antigen blocking agent to said cells wherein the mitosis associated nuclear antigen is about 47 kD and is capable of being a substrate for cdc2 kinase and wherein said blocking agent is a molecule capable of preventing said antigen from performing its role in mitosis.
  • Said blocking agent comprises a molecule capable of preventing said antigen from performing its role in mitosis including nucleic acid sequences which prevent transcription of the mitosis- associated antigen gene and IRMs specific for said antigen which by their actions thereby disrupt mitosis.
  • the blocking agent administered may be associated with the toxic agent discussed above.
  • the mitosis- associated nuclear antigen blocking agent comprises the nucleotide molecule of the invention or an IRM as described above.
  • the nucleotide molecule is in the form of an anti-sense or partial anti-sense sequence.
  • the nucleotide sequence of Fig. 4 inserted into a suitable vector such as pMT 4 SV neo to produce anti-sense sequences may be used.
  • the anti-sense sequence spans the promoter and/or start codon of said RMSA-1 and is in the region of 10 bp in length or any other convenient length.
  • the IRM is an antibody, more preferably a MAb specific for the antigen of the invention. Still more preferably the IRM is capable of blocking serine and threonine residues of the antigen such that phosphorylation is prevented.
  • the invention provides a method for disrupting or arresting mitosis in cells comprising administering an effective amount of an associated protein blocking agent to said cells.
  • the associated protein is of about 31 and is capable of phosphatase activity or is about 67 or 200 kD and is capable of being phosphorylated during mitosis and said associated protein is capable of association with the mitosis-associated antigen (described earlier) during mitosis.
  • Said agent comprises a molecule capable of preventing said associated protein from performing its usual function in mitosis and is preferably an IRM specific for one of the associated proteins.
  • the associated protein blocking agent is specific for the 31 kD associated protein.
  • the blocking agent is anti PP1 as herein described.
  • the present invention further extends to a method of treating cancer in a patient.
  • the invention provides a method of treatment of cancer cells in a patient which comprises administering an effective amount of a mitosis-associated nuclear antigen blocking agent or an associated protein blocking agent to said patient such that mitosis is arrested.
  • the mitosis-associated nuclear antigen is about 47 kD and is capable of being a substrate for cdc2 kinase and the associated protein is of about 31 and displays phosphatase activity or is about, 67 or 200 kD, and is capable of being phosphorylated during mitosis, wherein said associated protein is capable of association with said mitosis-associated nuclear antigen.
  • the blocking agent may be the same as that described above.
  • the blocking agent optionally comprising a toxic agent may be administered in combination with a pharmaceutically acceptable carrier or excipient by systemic administration, regional perfusion, topically or any other convenient means depending on the nature of the cancer being treated.
  • the invention provides a method of identification or localisation of dividing cells in a cell or tissue sample.
  • the present invention provides a method of identifying or localising dividing cells, particularly rapidly dividing cells, in sample which method comprises adding an effective amount of a mitosis-associated nuclear antigen binding agent specific for the mitosis-associated state of a mitosis-associated nuclear antigen and detecting the presence of binding to said antigen.
  • a mitosis-associated nuclear antigen binding agent specific for the mitosis-associated state of a mitosis-associated nuclear antigen and detecting the presence of binding to said antigen.
  • said binding agent is labelled with a detectable label as previously described.
  • the binding agent is specific for the antigen in its phosphorylated state.
  • the mitosis-associated nuclear antigen binding agent includes any agent which specifically binds the antigen when it is in its mitosis-associated state. This means the agent does not bind the antigen in cells not undergoing mitosis.
  • the agent includes an IRM, more preferably an antibody and most preferably a MAb specific for the mitosis- associated state of the antigen.
  • the method of identifying or localising dividing cells can be carried out using an effective amount of an associated protein binding agent specific for the mitosis-associated state of said associated protein in the manner described above.
  • the present invention also extends to a method of carrying out karyotype analysis.
  • the invention provides a method of arresting mitosis in cells so that the chromosomes can be made subject of a karyotype analysis, said method comprising administering an effective amount of a mitosis- associated nuclear antigen blocking agent to the cells.
  • an associated protein blocking agent can be used in the method of the invention to arrest mitosis.
  • the mitosis-associated nuclear antigen blocking agent is a nucleic acid molecule in the form of a full length or partial anti-sense sequence of any convenient size. Still more preferably the nucleic acid molecule spans the promoter and/or start sequence of RMSA-1 as shown in Fig. 4 herein.
  • the present invention also extends to methods of detecting and diagnosing autoimmune disease.
  • the invention provides a method of detecting autoimmune disease in a subject where an autoantibody directed to a protein of about 47 kD which protein is a substrate of cdc2 kinase is indicative of the presence of said disease, said method including contacting a sample from the subject likely to contain the autoantibody with an isolated preparation of the protein for a time and under conditions sufficient for a complex to form and detecting presence of said complex.
  • the invention also provides a method of diagnosing autoimmune disease in a patient where an autoantibody reactive with a protein of about 47 kD which protein is a substrate for cdc2 kinase is correlated with the presence of said disease, said method including contacting a sample suspected of containing the autoantibody from said patient with an isolated preparation of the protein for a time and under conditions sufficient for a complex to form and detecting said complex.
  • the autoimmune disease may be any disease wherein autoantibody is implicated in the disease process and may include diseases such as systemic lupus erythematosus, rheumatoid arthritis, schleroderma, polymyositis, dermatomyositis and mixed connective tissue disease.
  • the subject or patient may be a human or animal suspected of having the particular autoimmune disease under consideration.
  • the sample may be any body fluid which contains, or is likely to contain the autoantibody.
  • the sample is derived from blood, serum, tissue fluids.
  • the isolated preparation of the protein is preferably the protein according to the invention described earlier for which the autoantibody is specific.
  • the protein is RMSA-1 as hereinbefore described.
  • the protein is present on a support, more preferably a solid support.
  • Supports such as polymers, including nitrocellulose, paper, polyacrylamide, nylon, polystyrene, polyvinylchloride or polypropylene may be used.
  • the presence of the autoantibody-protein complex may be detected by an antiimmunoglobulin labelled with a label, reporter molecule such as protein A or other detector molecule capable of providing a detectable signal such as those described earlier.
  • Techniques such as ELISA, Western Blotting and RIA may be used to detect the complex.
  • kits comprising the reagents for karyotype or other analysis of cells.
  • the kit may be in compartmentalised form, said kit comprising a first compartment adapted to receive a mitosis-associated nuclear antigen blocking agent or an associated protein blocking agent and optionally at least one other compartment adapted to contain a detector means.
  • the present invention relates to a kit for detecting autoimmune disease in a patient wherein an autoantibody reactive with a protein of about 47 kD which protein is a substrate for cdc2 kinase, is correlated with said disease, comprising a first compartment adapted to act as a solid support for an isolated preparation of the protein wherein said first compartment is adapted to receive a sample from said patient suspected of containing said autoantibody, and at least one other compartment adapted to contain appropriate reagents and buffers for detecting a complex which may be formed by said autoantibody and said protein.
  • RMSA-1 localises to chromosomes of mitotic cells
  • the autoimmune serum from a patient with discoid lupus erythematous, reacted specifically with chromosomes of mitotic Hep-2 cells by immunofluorescence (Fig. 1). .
  • the autoantibody contained in the serum may be indicative of the presence of autoimmune disease, in particular lupus and other related autoimmune disease.
  • Cells in early prophase showed coarse speckled nuclear immunofluorescence corresponding to DNA patches stained by Hoechst DNA dye. Immunofluorescence of entire chromosomes persisted as cells progressed through mitosis from prophase to metaphase and anaphase, became considerably weaker at telophase and was lost at cytokinesis. Interphase cells were non-reactive. Similar immunofluoresence patters were seen in HeLa cells, mouse 3T3 fibroblasts, mouse L fibroblasts and monkey kidney COS cells (data not shown).
  • RMSA-1 is an evolutionarily conserved 47 kD nuclear protein
  • Fig. 2 A) Proteins (10 ⁇ g/lane), separated by 10% SDS-PAGE under reducing and non-reducing conditions and transferred to nitrocellulose membrane, were immunoblotted with human sera as described 12 . B) Cytoplasmic and nuclear subcellular fractions were derived as described 13 . C) Total HeLa cell lysates were processed for two-dimensional NEPHGE and immunoblotting as described 14 . D) HeLa cells were metaphase-arrested by 20 hr incubation with nocodazole (0.2 ⁇ g/ml) at 37°C.
  • Metaphase-arrested and interphase HeLa cells were radiolabelled with 0.5 mCi [ 35 S]-methionine/cysteine in methionine and cysteine-free medium or 1 mCi [ 32 P]- orthophosphate in phosphate-free medium for 8 hr at 37°C.
  • Labelled cells were extracted in lysis buffer (50 mM Tris-HCl, pH 8.0, lOmM EDTA, 150 mM NaCl, 1% NP40) containing 1 mM each of protease inhibitors PMSF, leupeptin, pepstain and aprotinin.
  • Cell extracts were immunoprecipitated with human sera as described 12 and analysed by 10% SDS-PAGE and autoradiography.
  • pJPZlL4 (1 ⁇ g) was linearised by Apal digestion and in vitro transcribed at 37°C for 1 hr using the T3 promoter sequence of pBluescript as per manufacturer's instructions (Promega). RNA transcripts were analysed in 1.0% agarose gels containing 2.2 M formaldehyde and in vitro translated using radiolabelled 35 S-methionine. The in vitro translated product was analysed by 10% SDS-PAGE, and immunoprecipitated with human sera as described 15 .
  • Methods A) Cells were harvested in 0.5 ml PBS/5% FCS, to which was added 500 ⁇ l of 10 ⁇ g/ml propidium iodide/0.02% Triton X-100 and 50 ⁇ l chicken red blood cells. Samples were filtered through a 50 ⁇ m nylon mesh and incubated with RNAse (10 ⁇ g/ml) on ice for 5 min in the dark. Samples were analysed using a FACSCAN and lysis II software. B) Cells were processed for immunoblotting as described in the legend to Fig. 2.
  • RMSA-1 is associated with 31, 67 and 200 kD molecules
  • RMSA-1 is phosphorylated during mitosis
  • the 47 kD RMSA-1 protein was phosphorylated only in metaphase- arrested cells (Fig. 2D, lane 4) and not in interphase cells (Fig 2D, lane 3).
  • Parallel immunoprecipitations with 35 S- methionine/cysteine-labelled interphase and metaphase cells confirmed that equivalent levels of RMSA-1 were present in the immunoprecipitates indicating that the lack of phosphoproteins in the interphase cells is not due to absence of protein (data not shown).
  • RMSA-1 phosphorylated RMSA-1 was immunoprecipitated by the autoimmune serum from HeLa cells, 10-13 hr following release from double thymidine block when most cells were in the G2/M phase of the cell cycle (Fig 3A); the phosphorylated 47 kD antigen was not immunoprecipitated from cells in the G0/G1 or S phases (data not shown).
  • RMSA-1 is phosphorylated only in mitosis.
  • the polypeptides of 31 kD, 67 kD and 200 kD which co-precipitate with RMSA-1 were also phosphorylated in mitosis (Fig. 2D, lane 4).
  • F A full length cDNA encode RMSA-1
  • Reco binants (IO 6 ) from a human hepatoma Hep-G2 ⁇ zap cDNA expression library (Stratagene) were screened with the autoimmune serum (diluted 1:50) 23 .
  • a single clone ( ⁇ JPL4) was isolated, plaque-purified, and in vivo excised to obtain the pBluescript plasmid, pJPL41.6; pJPL41.6 was derived from pJPL4 by EcoR 1 digestion and subcloning.
  • two overlapping oligonucleotides (5' -CCATGGGAGATGGCCCAACAAATGTTTAAA-3' and
  • 5' -CCCAGCTCTTTTACTTTTTAAACATTTG-3' were designed from the 5' sequence of pJPL41.6, 32 P-labelled using the standard filling reaction of Klenow polymerase and used to screen the library. Two clones pJPZl and pJPZ2 were identified, purified and sequenced. The full length cDNA encoding RMSA-1 was constructed by ligating inserts from pJPZl and pJPL41.6 and subcloning into pBluescript (KS-) vector (Stratagene). Plasmid DNA was transformed into MV1190 cells as described 24 .
  • KS- pBluescript
  • RNA (10 ⁇ g) isolated from HeLa cells as described 25 was separated by 1% agarose gel electrophoresis containing 2.2 M formaldehyde and transferred to Hybond-N + nylon membrane (Amersham). The membrane was incubated with prehybridisation solution (7% SDS, 6X SSC, 1% EDTA and 0.25% w/v skim milk) at 65°C for 2 hr and hybridised overnight at 65 "C with 32 P- labelled RMSA-1 cDNA (6 X IO 6 ) cpm/ml in prehybridisation solution. Membranes were washed in 0.1X SSC/0.1% SDS at 65 ⁇ C and exposed to Fuji X-ray film.
  • DNA sequence was obtained by the dideoxy nucleotide termination method 26 , double stranded sequencing by the method of Chen and Seeburg 27 and single stranded sequencing using a T7 DNA polymerase, deaza- GTP sequencing kit (Promega). Nucleic acid and protein sequences were analysed using the University of Wisconsin Genetic Computer Group Sequence Analysis Software Package. Data bases searched were current, weekly-updated Genebank, EMBL and Nucleic acid reference banks.
  • the plasmid pJPL41.6 was derived by immunoscreening a human ⁇ zap hepatoma cDNA library and pJPZl was obtained by 32 P-oligonucleotide hybridisation to the same library (Fig. 4A). The two plasmids were ligated to produce pJPZlL4. Blot analysis of HeLa cell RNA with pJPZlL4 revealed a single mRNA species of about 3kb (Fig. B). pJPIL4 appeared to be a full length clone with a 774 bp 5' untranslated region, an open reading frame of 1254 bp and a long 3' untranslated region (Fig. 4C).
  • the open reading frame encodes a novel protein of 418 amino acids with a predicted molecular weight of 47.9 K and a neutral pi of 6.6.
  • the deduced amino acid sequence of RMSA-1 shows 3 ST/P consensus motifs for phosphorylation by cdc2 kinase and a run of 10 basic lysines and KKKQRK sequences resembling conserved nuclear localisation signals 28 .
  • the predicted molecular weight of 47.9 K and pi of 6.6 for the protein encoded by pJPZlL4 correspond to the biochemical characteristics of RMSA-1.
  • a bacterial fusion protein produced by pJPL41.6 cDNA which incorporates a partial amino acid sequence of RMSA-1 was specifically reactive with the autoimmune serum (Fig 2E, lane 4).
  • the molecular weight of the fusion protein is consistent with the predicted combined Mr of the 3 kD fragment of ⁇ -galactosidase and the partial protein encoded by the incomplete RMSA-1 cDNA clone.
  • the plasmid pMT 4 SV neo contains four copies of a synthetic mouse metallothionein-1 promoter element, a G418 resistance gene and a SV40 T antigen gene 29 - 30 .
  • the SV40 T antigen gene was replaced with full length RMSA-1 cDNA to generate both sense and anti-sense constructs.
  • Mouse L cells were transfected with plasmid DNA by the calcium phosphate/15% glycerol shock procedure and selected with G418 (300 ⁇ g/ml). Isolated colonies were picked and expanded into stable cell lines. Sense and anti-sense transfected cells were induced by 100 ⁇ M ZnCl 2 .
  • affinity-purified autoantibody For introduction of affinity-purified autoantibody into mouse L cells, 5 x IO 6 cells were electroporated at 250 ⁇ F and a constant voltage of 0.45 kV (Biorad). The cells were held in a cuvette with a path length of 0.8 cm holding 0.8 ml PBS/10 mM MgCl 2 containing 0.5 mg/ml affinity purified anti-RMSA-1 autoantibody or protein G- purified human IgG. Following electroporation and 10 min incubation on ice, the cells were washed and plated in complete tissue culture medium. Non-adherent dead cells were removed at 3 hr by a change of culture medium. Cell morphology was monitored over 24 hr and entry of antibody was confirmed by direct immunofluorescence and flow cytometry using FITC-labelled rabbit anti-human immunoglobulin.
  • RMSA- 1 Two strategies we adopted to study the function of RMSA- 1, namely an anti-sense mRNA approach and the introduction of affinity-purified anti-RMSA-1 autoantibody into cells by electroporation.
  • anti-sense strategy a construct was made that allowed production of RMSA-1 anti-sense mRNA in stably transfected cells. As inhibition of RMSA-1 gene expression may be lethal, the production of anti-sense transcripts was regulated with MT 4 , a synthetic mouse metallothionein promoter responsive to heavy metals and which had minimal basal activity 29 - 30 .
  • the anti-sense and sense cDNA, with the entire open reading frame encoding RMSA-1 were cloned into the pMT 4 vector. The construct was transfected into mouse fibroblast Ltk- cells and stable cell lines were established.
  • the floating cells observed at 24 hr post-zinc induction, were harvested and cytocentrifuged for microscopic examination. Phase contrast microscopy and staining with Hoechst DNA dye showed that most of the anti-sense transfected cells were arrested in mitosis, displaying condensed chromosomes typical of cells in prometaphase (Fig. 5B, panel a). These mitotic-arrested cells did not react with the anti- RMSA-1 antibody by immunofluorescence or by immunoblotting (data not shown) indicating that the anti-sense mRNA had inhibited RMSA-1 expression. Flow cytometric analysis of the zinc-treated sense transfected cells (Fig.
  • RMSA-1 is required for mitotic spindle assembly
  • cytocentrifuged preparations of transfected or electroporated cells were reacted by immunofluorescence with autoantibodies to centromeres, centrosomes, and nuclear lamins 31 and with a rabbit antibody to tubulin.
  • centrosomes had divided (Fig. 6b)
  • centromeres had formed and the nuclear lamina had broken down.
  • controls, including sense-transfected cells displayed typical mitotic spindles (Fig. 6a)
  • the mitotic spindle had failed to form in the anti- sense transfected cells induced with zinc (Fig. 6b).
  • the work described herein identifies a novel and conserved protein associated with condensed chromosomes which is phosphorylated at mitosis and whose presence is necessary for assembly of the mitotic spindle.
  • the localisation of this protein to chromosomes throughout mitosis is clearly distinct from that of "chromosomal passenger" proteins, a group of "migratory” proteins associated with chromosomes during prometaphase and metaphase and which relocate to the midzone of the mitotic spindle at anaphase 32 .
  • the chromosomal localisation of RMSA-1 also sets it apart from known centromere autoantigens, which have distinct immunofluorescence localisation patterns in interphase and in mitotic cells 33 .
  • the cdc2 serine/threonine protein kinase plays a pivotal role in the regulation of the eukaryotic cell cycle 2 - 3 .
  • the kinase active at the G2/M transition, is thought to phosphorylate key molecules which mediate events accompanying mitosis.
  • only a few candidate substrates for the kinase linked to mitotic events have been established. These include phosphorylation of nuclear lamins which regulate nuclear lamina breakdown 34 " 36 and phosphorylation of cytoskeletal proteins vimentin and caldesmon associated with reorganisation respectively of intermediate filaments 37 and microfilaments 38 .
  • RMSA-1 appears to serve as a physiological substrate for the cdc2 kinase since it is phosphorylated only in mitotic cell in vivo and contains the kinase consensus motifs. This is further supported by the observation that immunoprecipitated RMSA-1 from interphase cells can be phosphorylated by cdc 2 kinase (immunoprecipitated from metaphase-arrested cells) in the presence of ⁇ 3 PATP; whereas RMSA-1 immunoprecipitated from metaphase-arrested cells cannot be phosphorylated by the kinase, suggesting saturation of the kinase phosphorylation sites during mitosis (Yeo, J.P et al, manuscript in preparation).
  • RMSA-1 immunofluorescence localisation of RMSA-1 to condensed chromosomes throughout mitosis suggests that one possible role for this molecule may be in the regulation of chromosomal condensation.
  • inhibition of the autoantigen by RMSA-1 anti-sense mRNA or by autoantibody did not prevent chromosomal condensation a role for RMSA-1 in chromosomal condensation has not been excluded as there may be fail-safe mechanisms for the preservation of this process.
  • the other molecules associated with RMSA-1 may have similar and overlapping roles in chromosomal condensation.
  • RMSA-1 is required for assembly of the mitotic spindle necessary for chromosomal segregation.
  • RMSA-1 is phosphorylated in mitosis suggests that it is the phosphorylated form which is active in this process.
  • a number of kinesin-like microtubule-"motor" proteins have recently been shown to affect the organisation of the mitotic spindle 39 " 2 .
  • RMSA-1 does not visibly associate with any component of the mitotic spindle poles or spindle proper, and its amino acid sequence shows no discernible sequence similarity to any of the known microtubule-"motor" proteins 1 .
  • RMSA-1 must affect mitotic spindle assembly by a different mechanism.
  • the phosphorylated chromosomal protein acting either alone or in concert with associated phosphorylated polypeptides, may be responsible for regulating the dynamics of microtubule depolymerisation and polymerisation necessary for conversion of microtubule arrays in interphase to the mitotic spindle in metaphase 1,2 .
  • Mitotic cells were prepared by single thymidine block followed by nocodazole (NO) treatment which prevents mitotic spindle formation and arrests the synchronised cells in a pseudo-metaphase state. After 18 h treatment with 0.1 ⁇ g/ml NO, mitotic cells were harvested by shake-off. Hoechst staining showed that 90% of these cells were in mitosis displaying condensed chromosomes. For metabolic labelling with 0.5 mCi [ 35 S]-methionine/cysteine, interphase and mitotic cells were starved for 1 h and labelled for 8 h in the presence of NO.
  • NO nocodazole
  • Affinity purified anti-RMSA-1 autoantibody or NHS-IgG was bound to protein A sepharose beads in lysis buffer. Nuclear extracts were prepared and used for immunoprecipitation as described 44 .
  • unlabelled immunoprecipitates PP1 or PP2A were transferred to nitrocellulose and immunoblotted as described 44 with antibody to type 1 protein phosphatase (PP1) traced with 1:100 dilution of NRP-labelled anti-rabbit antibody (DAKO). These antibodies were generated in rabbits to a synthetic peptide corresponding to the C-terminal 316-330 aminoacid residues of human PPl ⁇ (UBI, USA), b.
  • Immunoprecipitates were ' washed in low salt buffer containing 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 0.01% Brij 35, 0.5 % 2-mercaptoethanol.
  • the immunoprecipitates were resuspended in 20 ⁇ l of buffer. After incubation in buffer at 30°C for 30 min, monoiodinated microcystine-YR (1000 cpm) was added and incubated at room temperature for 30 min. The samples were boiled in SDS-PAGE sample buffer before electrophoresis. The gel was fixed, dried and exposed to autoradiography. Catalytic subunits of PPl and PP2A purified from rabbit skeletal muscle and monoiodinated microcystin were gifts from P. Cohen.
  • Fig. 5 a.
  • the 31 K molecule of the RMSA-1 is immunoreactive with antibody to type 1 protein phosphatase.
  • Immunoprecipitates of the RMSA-1 complex in nuclear extracts of interphase (I, lane 3) and mitotic (M, lane 4) HeLa cells, metabolically labelled with [ 35 S]methionine/cysteine showed the 31 K, 47 K, 67 K and 200 K components.
  • Control immunoprecipitates with protein G- purified normal human IgG (NHS-IgG) were also conducted (lanes 1 and 2).
  • the 31 K RMSA-1 associated molecular was reactive with microcystine. Immunoprecipitates of RMSA-1 complex from interphase HeLa cells, incubated with 125 I labelled microcystin-Yr, showed reactivity with a 31 K (lane 2) molecular with co-migrates with PPl ⁇ (lane 1).
  • RMSA-1 immunoprecipitates were washed in 50 mM Tris-HCl, pH 7.5, 30mM NaCl, 0.1% 2-mercaptoethanol, 0.5mM PMSF before resuspending in the phosphatase buffer.
  • 32 P- labelled glycogen phosphorylase a (containing 1.0 mol phosphate/mol) was prepared by phosphorylation with phosphorylase kinase according to the manufacturer's instructions (Gibco).
  • assays of immunoprecipitates were carried out at 1/10 dilution (to obtain phosphatase activity within a linear range) in phosphatase buffer containing 50mM Tris-HCl, O.lmM EGTA, 0.1% (v/v) 2-mercaptoethanol, 25 mg/ml aprotinin, 25 mg/ml leupeptin and 0.5mM PMSF.
  • 1/10 dilution of immunoprecipitates gave a phosphatase concentration of 0.02 ⁇ g/ml (2 ng) and a specific phosphatase activity of 1-2.8 J/min/ ⁇ l.
  • the phosphatase assays were carried out in duplicates at 30°C in 60 ⁇ l volume of 15 min 57 . Assays were terminated with 20% trichloroacetic acid and analysed using a ⁇ -counter. Base-line activity, obtained by replacement of the protein phosphatase with the assay buffer, were subtracted from those obtained in the presence of phosphatase. Successful dephosphorylation of each substrate was taken as 30% or less as within this range the release of phosphate was linear with time. 1U phosphatase activity was that amount of enzyme which release 1 ⁇ mol phosphate from glycogen phosphorylase a in 1 min. b.
  • inhibitor 1 okadaic acid and microcystin-LR were standardized using the catalytic subunits of protein phosphatase 1, with phosphorylase a as substrate. Using catalytic subunits at ⁇ 0.1U/ml, inhibitor 1 completely blocked PP-1 activity at 1 uM but did not affect PP2A activity. Okadaic acid at 5 nM completely inhibited PP2A but did not affect PPl, whereas 1 ⁇ M okadaic acid blocked both PPl and PP2A. Microcystin-LR inhibited both PPl and PP2A with similar potency at 10 nM.
  • PPl activity was taken as that inhibited by 1 ⁇ M inhibitor 1 or that inhibited by 1 ⁇ M okadaic acid but not by 5 nM okadaic acid.
  • PP2A activity was taken as that inhibited by 5 nM okadaic acid but not 1 ⁇ M inhibitor 1.
  • Fig. 6 Protein phosphatase activity of RMSA-1 complex. Interphase and mitotic nuclear extracts were immunoprecipitatedwith anti-RMSA-1 autoantibody or with control NHS-IgG. Phosphatase assays were performed using 32 P-phosphorylase a and histone HI as substrates. Phosphatase activity (see Bars) as 32 P counts per minute (cpm) released in the assay were calculated. b. Effect of inhibitors on phosphatase activity.
  • the RMSA-1 immunoprecipitates were incubated at 30 ⁇ C for 15 min with 1 ⁇ M inhibitor I ( ), 5 nM okadaic acid ( ), 1 ⁇ M okadaic acid ( ), 10 nM microcystin ( ). After incubation, the immunoprecipitates were tested for phosphatase assay using 32 P-phosphorylase a as substrate. A control incubation without phosphatase inhibitors ( ) was also conducted. The results of inhibition by inhibitor 1 and okadaic acid indicate that the phosphatase activity in the immunoprecipitates is PPl-like.
  • Active cdc2 kinase was prepared from mitotic HeLa cells by immunoprecipitation with mouse monoclonal anti-cdc2 antibodies (Santa Cruz), as described 58 . Briefly, HeLa cells from 80% confluent tissue culture flasks were lysed in 1 ml of RIPA buffer containing 9.1M dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 mM Na 3 P0 4 and 50 mM NaF.
  • Proteins were immunoprecipitated with 1 ⁇ g of antisera coupled to protein A-sepharose beads (Pharmacia) in RIPA buffer. Immunobilized immunoprecipitates were washed thrice in RIPA buffer and pellets were resuspended in 20 ⁇ l of protein kinase assay buffer (50mM Tris-HCl, pH 8, lOmM MgCl 2 , 20 mM EGTA, 1 mM dithiothreitol, 4 mM ⁇ - glycerophosphate, 25 ⁇ g/ml leupeptin, 0.5 mM phenylmethy1sulfonyl fluoride).
  • protein kinase assay buffer 50mM Tris-HCl, pH 8, lOmM MgCl 2 , 20 mM EGTA, 1 mM dithiothreitol, 4 mM ⁇ - glycerophosphate, 25 ⁇ g/m
  • the RMSA-1 complexes coupled to protein A-sepharose beads were washed twice in the kinase assay buffer and resuspend in 20 ⁇ l of kinase assay buffer.
  • 20 ⁇ l of purified cdc2 kinase, resuspended in assay buffer containing 50 ⁇ M ATP and preincubated at 30 ⁇ C for 15 min was added to the RMSA-1 complexes in the presence of 10 ⁇ Ci of [ ⁇ - 32 P]ATP (Bresatec) for 30 min at 30°C.
  • the reaction was stopped by washing the beads in RIPA buffer and the complexes analysed by SDS- PAGE/autoradiography. b,c.
  • the phosphorylated immuno ⁇ precipitates were subject to SDS-gel electrophoresis and electroblotted onto polyvinylidene difluoride membrane for phosphoamino acid 39 , or nitrocellulose for phosphopeptide analysis 60 .
  • the areas on the membrane containing the 32 P- labelled RMSA-1 protein (as located by autoradiography) were excised.
  • the 32 P-labelled protein bound to Immobilon membrane was hydrolysed with 6 M HCI for 1 h at 100 ⁇ C.
  • the hydrolysates were dried in vacuo,and dissolved in 5 ⁇ l of pH1.9 buffer (formic acid:acetic acid:water, 50:156:1794).
  • Samples and phosphoamino acid standards (phosphotyrosine, phosphoserine and phosphothreonine; Sigma) were separated by two dimensional electrophoresis, first dimension using pH 1.9 buffer and second dimension using pH 3.5 buffer (acetic acid: pyridine:water, 100:10:1890) on thin-layer ehromatography plates (Merck) 23 , using an HTLE-7000 apparatus (C.B.S. Scientific Company Inc. ).
  • tryptic phosphopeptide mapping the 32 P-labelled protein on nitrocellulose was digested with 10 ⁇ g TPCK-treated trypsin (promega) in 50mM ammonium bicarbonate buffer, pH 8 overnight at 37 ⁇ C.
  • the phosphorylated complex was washed in 50 mM Tris/HCl, pH 7.5, 30mM NaCl, 0.1% 2-mercaptoethanol, 0.5 mM PMSF and resuspended in 10 ⁇ l of phosphatase buffer, e.
  • the activated complex was preincubated with 1 ⁇ M inhibitor, 5 nM okadaic acid, or 1 ⁇ M okadaic acid for 15 min at 30 ⁇ C.
  • the complex was then tested for phosphatase activity. Immunoprecipitates with NHS-IgG and PPl ⁇ were included as controls.
  • Fig 7 a. Autoradiograph of RMSA-1 immunoprecipitates of nuclear phosphoproteins.
  • HeLa cells interphase and metaphase-arrested labelled in vivo with 32 P- orthophosphate were immunoprecipitated with anti-RMSA-1 autoantibody (lanes 3 and 4) or with NHS-IgG (lanes 1 and 2).
  • RMSA-1 immunoprecipitated from interphase (lane 5) but not from mitotic cells (lane 6) are phosphorylated in vitro with active cdc2 kinase.
  • the active cdc 2 kinase was immunoprecipitated with a mouse monoclonal antibody to cdc2 kinase.
  • the RMSA-1 complex phosphorylated in the presence of cold ATP was also removed at the same time intervals, immunoprecipitates washed twice in phosphatase buffer and tested for phosphatase activity, c.
  • HeLa cytosolic interphase extracts were prepared as described 62 . Labelled and unlabelled immunoprecipitates were incubated with 0.3 mg/ml of interphase extracts for 15 min at 30°C. In separate experiments, okadaic acid or inhibitor 1 was added to the interphase extracts for 15 min at 30°C before incubating with the immunoprecipitates. The labelled complex was then analysed by SDS-PAGE. d. The unlabelled complex, treated as described in (c), was tested for phosphatase activity.
  • Fig. 8 a. RMSA-1, phosphorylated in vitro with cdc2 kinase, in the presence of [ ⁇ 32 P]-ATP was incubated with 5 mM EDTA at 30°C. At time intervals indicated in Fig. 8, aliquots were removed and analysed by SDS-PAGE and autoradiography. b. Unlabelled phosphorylated RMSA-1 treated with 5 mM EDTA were also removed at the same time intervals for phosphatase assay. c. Dephosphorylation of RMSA-1 by interphase extracts for exponentially growing HeLa cells.
  • RMSA-1 phosphorylated in vitro by cdc2 kinase, in the presence of [ ⁇ - 32 P]-ATP was added to interphase extracts at 0.3 ⁇ g/ml final concentration. After 15 min incubation at 30°C, gel sample buffer was added and the phosphorylation state of RMSA-1 complex was assessed by SDS-PAGE (lane 2). Experiments were also carried out in the presence of 0.5 ⁇ M okadaic acid (lane 4), 1 ⁇ M okadaic acid (lane 5) or 1 ⁇ M inhibitor 1 (lane 3). Controls included incubation of phosphorylated RMSA-1 in buffer alone (lane 1). d.
  • the anti-PPl antibody immunoblotted the 31 K protein of the RMSA-1 complex from interphase (Fig. 1, lane 7) and metaphase-arrested cells (Fig. 1, lane 8).
  • the immunoblotted 31 K protein co-migrated with purified PPl ⁇ (Fig. 1, lane 9).
  • the identity of the associated phosphatase as a catalytic subunit of a Type 1 phosphatase is further supported by labelling of the 31 K component in the RMSA-1 immunoprecipitates with iodinated microcystine-YR (Fig. 5, panel B, lane 2).
  • the labelled 31 K molecule also co-migrated with PPl ⁇ (lane 2).
  • RMSA-1 associated phosphatase was activated during mitosis.
  • immunoprecipitates from interphase and mitotic extracts were tested for phosphatase activity using 3 P-labelled phosphorylase ⁇ or histone HI as substrates.
  • the RMSA-1 complex from mitotic cells showed a dramatic increase in phosphatase activity, compared to that from interphase cells.
  • the RMSA-1 complex from mitotic cells is sensitive to inhibition by 1 ⁇ M inhibitor I and 1 uM okadaic acid but insensitive to 5 nM okadaic acid (Fig. 6, panel B).
  • PP2A is inhibited by nanomolar concentrations of okadaic acid but insensitive to inhibitor 1, we conclude that the RMSA-1 associated phosphatase, activated during mitosis, has characteristics compatible with a type I phosphatase.
  • RMSA-1 is phosphorylated only during mitosis and contains consensus motifs for phosphorylation by cdc2 kinase 44 , we tested whether cdc2 kinase can phosphorylate
  • RMSA-1 in vitro.
  • Activated cdc2 kinase was purified from mitotic HeLa cells by immunoaffinity ehromatography using monoclonal antibody to cdc2 kinase.
  • Immunoprecipitated RMSA-1 complex was incubated with the purified active cdc2 kinase in a kinase reaction containing [ ⁇ 32 P]-ATP (Fig. 7, panel A, lanes 5 and 6). With the exception of the 31 K protein, the other proteins of the RMSA-1 complex from the interphase extract were phosphorylated. The proteins in the RMSA-1 complex from metaphase extracts were not phosphorylated suggesting saturation of the phosphorylation sites in mitosis.
  • RMSA-1 As RMSA-1 is activated by phosphorylation on entry into mitosis and is not phosphorylated in interphase 44 , clearly it has to be dephosphorylated and inactivated to exit mitosis.
  • the RMSA-1 complex was immunoprecipitated from interphase nuclear extracts, phosphorylated in vitro by cdc2 kinase and the kinase inactivated with EDTA. There was no decrease in phosphorylation of RMSA-1 up to 15 min incubation (Fig * 8, panel A, lanes 1 to 5). Cold immunoprecipitates also showed no inhibition of phosphatase activity (Fig. 8, panel B).
  • the in vivo phosphorylated RMSA-1 complex was incubated with interphase and nuclear extracts rich in phosphatase activity. After 10 min incubation in interphase extracts, there was a marked loss of about 90% of radioactivity from the phosphorylated RMSA-1 complex (Fig. 8, panel C, lane 2). The decrease in radioactivity was not inhibited by 1 ⁇ M inhibitor 1 (lane 3) but by 0.5 ⁇ M and 1 ⁇ M okadaic acid (lane 5). Similarly, the phosphatase activity of the RMSA-1 complex was completely abolished by incubation with interphase extracts.
  • Type 1 protein phosphatase High levels of type 1 protein phosphatase are present in nuclei 47-48 , displaying increased activity during mitosis 49 .
  • Type 1 phosphatases are required for mitotic exit in yeast 50,51 , Drosophila 52 and mammalian cells 53 .
  • the type 1 phosphatase catalytic subunits are evolutionarily highly conserved 46 .
  • a flowering plant type 1 phosphatase can rescue a yeast mutant phosphatase 54 .
  • a yeast protein, sds22+ has been suggested to be a regulatory subunit of a yeast type 1 protein phosphatase necessary for mitotic spindle formation and exit from mitosis 55 .
  • RMSA-1 is similar to sds 22+ because it has leucine-rich sequences 44 ' 56 , it differs from the yeast protein in that it has consensus motifs for cdc 2 kinase and seems to be a physiological substrate for this kinase. Further, it appears that RMSA-1 is regulated by phosphorylation mediated by cdc2 kinase and dephosphorylation mediated by a type 2A phosphatase. In turn, these post- translational modifications of RMSA-1 respectively activate or inactivate the catalytic subunit of the phosphatase. These events are coupled to successful progression through mitosis 44 .
  • RMSA-1, activated by cdc2 kinase is a regulatory subunit of a chromosome-associated type 1 phosphatase catalytic subunit.
  • Cicuttini, F.M., Martin, M. Salvaris, E., Ashman, L., Begley, C.G., Novotny, J., Maher, D., & Boyd, A.W. Blood

Abstract

Nouvelle molécule d'acide nucléique isolée codant un antigène nucléaire associé à la mitose, ledit antigène d'environ 47 kD pouvant être phosphorylé pendant la mitose et constituer un substrat pour la cdc2 kinase, ou dérivé, variante, homologue, analogue ou fragment de ladite molécule d'acide nucléique qui est fonctionnellement en rapport avec ladite molécule d'acide nucléique. La présente invention concerne également une protéine isolée comportant un antigène nucléaire associé à la mitose, d'environ 47 kD, pouvant être phosphorylé pendant la mitose et constituer un substrat pour la cdc2 kinase, d'autres protéines associées et les utilisations desdites molécules d'acide nucléique et protéines, ainsi que des méthodes de diagnostic et de traitement de maladies et des préparations pharmaceutiques.
PCT/AU1994/000158 1993-03-31 1994-03-31 Sequence de nucleotides codant une proteine chromosomique, proteines chromosomiques associees et leurs utilisations WO1994023029A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997041153A1 (fr) * 1996-05-02 1997-11-06 Cold Spring Harbor Laboratory Genes regulant la replication de l'adn
WO1999013083A2 (fr) * 1997-09-05 1999-03-18 Cropdesign N.V. Methode et dispositif de modulation de proteines de cycle cellulaire vegetal et leur utilisation dans la regulation de la croissance de cellules vegetales
US6074819A (en) * 1996-05-02 2000-06-13 Cold Spring Harbor Laboratory DNA replication-regulating genes
US6350747B1 (en) 1999-03-04 2002-02-26 Glaxo Wellcome Inc. 3-(anilinomethylene) oxindoles
US6361954B1 (en) 1996-05-02 2002-03-26 Cold Spring Harbor Laboratory Methods of immunoassay for human CDC6
US6369086B1 (en) 1997-09-05 2002-04-09 Smithkline Beecham Corporation Substituted oxidole derivatives as protein tyrosine and as protein serine/threonine kinase inhibitors
US6492398B1 (en) 1999-03-04 2002-12-10 Smithkline Beechman Corporation Thiazoloindolinone compounds
US6620818B1 (en) 2000-03-01 2003-09-16 Smithkline Beecham Corporation Method for reducing the severity of side effects of chemotherapy and/or radiation therapy
US6624171B1 (en) 1999-03-04 2003-09-23 Smithkline Beecham Corporation Substituted aza-oxindole derivatives

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CELL, Volume 58, 1989, pages 485-497, BOOHER R. et al., "The Fission Yeast cdc2/cdc13/suc1 Protein Kinase: Regulation of Catalytic Activity and Nuclear Localization". *
CELL, Volume 60, (1990), pages 791-801, PETER M. et al., "Identification of Major Nucleolar Proteins as Candidate Mitotic Substrates of CaC2 Kinase". *
MOLECULAR BIOLOGY OF THE CELL, Volume 3, 1992, pages 687-698, WALKER D.H. et al., "Multiple Roles for Protein Phosphatase 1 in Regulating the Xenopus Early Embryomic Cell Cycle". *
NATURE, Volume 367, 20 January 1994, pages 288-291, YEO J.P. et al., "A New Chromosomal Protein Essential for Mitotic LSpindle Assembly". *
THE JOURNAL OF CELL BIOLOGY, Volume 120, No. 3, (1993), pages 613-624, HARTL P. et al., "Mitotic Repression of Transcription In Vitro". *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6074819A (en) * 1996-05-02 2000-06-13 Cold Spring Harbor Laboratory DNA replication-regulating genes
US5851821A (en) * 1996-05-02 1998-12-22 Cold Spring Harbor Laboratory DNA Replication-regulating genes
WO1997041153A1 (fr) * 1996-05-02 1997-11-06 Cold Spring Harbor Laboratory Genes regulant la replication de l'adn
US6361954B1 (en) 1996-05-02 2002-03-26 Cold Spring Harbor Laboratory Methods of immunoassay for human CDC6
US6387919B1 (en) 1997-09-05 2002-05-14 Glaxo Wellcome Inc. Substituted oxindole derivatives as protein tyrosine kinase and as protein serine/threonine kinase inhibitors
WO1999013083A3 (fr) * 1997-09-05 1999-05-06 Cropdesign Nv Methode et dispositif de modulation de proteines de cycle cellulaire vegetal et leur utilisation dans la regulation de la croissance de cellules vegetales
US6369086B1 (en) 1997-09-05 2002-04-09 Smithkline Beecham Corporation Substituted oxidole derivatives as protein tyrosine and as protein serine/threonine kinase inhibitors
WO1999013083A2 (fr) * 1997-09-05 1999-03-18 Cropdesign N.V. Methode et dispositif de modulation de proteines de cycle cellulaire vegetal et leur utilisation dans la regulation de la croissance de cellules vegetales
US6541503B2 (en) 1997-09-05 2003-04-01 Smithkline Beecham Corporation Substituted oxindole derivatives as protein tyrosine kinase and as protein serine/threonine kinase inhibitors
US7105529B2 (en) 1997-09-05 2006-09-12 Smithkline Beecham Corporation Substituted oxindole derivatives as protein tyrosine and as protein serine/threonine kinase inhibitors and compositions and methods of treating chemotherapy and radiation therapy side effects
US6350747B1 (en) 1999-03-04 2002-02-26 Glaxo Wellcome Inc. 3-(anilinomethylene) oxindoles
US6492398B1 (en) 1999-03-04 2002-12-10 Smithkline Beechman Corporation Thiazoloindolinone compounds
US6624171B1 (en) 1999-03-04 2003-09-23 Smithkline Beecham Corporation Substituted aza-oxindole derivatives
US6815439B2 (en) 1999-03-04 2004-11-09 Smithkline Beecham Corporation Substituted aza-oxindole derivatives
US6818632B2 (en) 1999-03-04 2004-11-16 Smithkline Beecham Corporation 3-(anilinomethylene)oxindoles
US7129253B2 (en) 1999-03-04 2006-10-31 Smithkline Beecham Corporation Compounds
US6620818B1 (en) 2000-03-01 2003-09-16 Smithkline Beecham Corporation Method for reducing the severity of side effects of chemotherapy and/or radiation therapy

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