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WO2006034300A2 - Kinases greatwall et leurs utilisations - Google Patents

Kinases greatwall et leurs utilisations Download PDF

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Publication number
WO2006034300A2
WO2006034300A2 PCT/US2005/033717 US2005033717W WO2006034300A2 WO 2006034300 A2 WO2006034300 A2 WO 2006034300A2 US 2005033717 W US2005033717 W US 2005033717W WO 2006034300 A2 WO2006034300 A2 WO 2006034300A2
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Prior art keywords
greatwall
polypeptide
seq
expression
activity
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WO2006034300A3 (fr
Inventor
Michael L. Goldberg
Jiangtao Yu
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Cornell Research Foundation Inc
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Cornell Research Foundation Inc
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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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the invention relates to kinase polypeptides and polynucleotides and uses thereof.
  • chromosomes DNA replication must be completed: for example, Drosophila mutant in DNA replication factor genes show defects in chromosome condensation (Krause et al., MoI. Cell. Biol. 21:5156-5168, 2001; Pflumm and Botchan, Development 128:1697- 1707, 2001).
  • cyclin B- CDK is inactive and stays in the cytoplasm during these early condensation stages (Rieder and Cole, J. Cell Biol. 142:1013-1022, 1998; Pines and Rieder, Nat. Cell Biol. 3:E3-E6, 2001). Later aspects of chromosome condensation do require cyclin B- CDK. Late in prophase, the nuclear accumulation and activation of cyclin B-CDKl triggers nuclear envelope breakdown (NEB), and also mediates subsequent chromosome condensation, in particular, cyclin B-CDKl phosphorylates and activates the 13S condensin complex (Kimura et al, Science. 282:487-490, 1998; Sutani et al., Genes Dev. 13:2271-2283, 1999).
  • NEB nuclear envelope breakdown
  • Cdc2 the major driver of mitosis, is regulated both by the synthesis and degradation of its cyclin B component, and by the control of Cdc2 through phosphorylation (reviewed by Tunquist and Mailer, Genes Dev. 17:683-710, 2003).
  • Cdc2 function requires phosphorylation by CDK-activating kinase, but Cdc2 is also negatively regulated by inhibitory phosphorylations on Thrl4 and Tyrl5.
  • the kinases responsible for these inhibitory phosphorylations are Weel and Mytl, while the phosphatase Cdc25 removes these phosphates and thus activates Cdc2.
  • the regulators of Cdc2 are themselves controlled by phosphorylations.
  • the MAPK pathway adds inhibitory phosphorylations to Mytl and Weel (Peter et al.,
  • Polo-like kinase phosphorylates and thus activates Cdc25 (Kumagai and Dunphy, Science 273:1377-80, 1996).
  • Mytl may also be a substrate for Polo-like kinase (Nakajima et al., J Biol Chem. 278:25277-80, 2003).
  • the MAPK pathway and Polo-like kinase are themselves controlled by Cdc2-cyclinB (Abrieu et al., J Cell Sci. 111:1751-57, 1998; Abrieu et al., J Cell Sci. 114:257-67, 2001), these components formally constitute a positive feedback loop that supports high MPF levels during mitosis (Pomerening et al., Nat Cell Biol. 5:346-51 5 .2003).
  • the present invention features polynucleotides and polypeptides that regulate mitosis.
  • a novel Drosophila gene greatwall, that is required for proper chromosome condensation.
  • the Greatwall protein is conserved in insects and vertebrates, and contains a kinase domain bifurcated by a long stretch of unrelated amino acids.
  • Greatwall kinase is found in the nuclei of interphase cells, but is distributed throughout mitotic cells.
  • Mitotic chromosomes in greatwall mutants are notably undercondensed in the euchromatin.
  • condensin, phosphorylated histone H3, and topoisomerase II are recruited to these abnormally condensed chromosomes.
  • NEB mutant cells showed major delays in cell cycle progression. The period of chromosome condensation prior to nuclear envelope breakdown (NEB) was much longer than normal. After NEB mutant cells displayed a delay in metaphase that is dependent on the spindle checkpoint, hi addition, Greatwall is a necessary component of the autoregulatory loop that triggers mitosis by blocking inhibitory phosphorylations on Cdc2.
  • the invention features an isolated polynucleotide selected from the group consisting of: a) a polynucleotide encoding a Greatwall polypeptide including an amino acid sequence selected from the group consisting of amino acid sequences that are substantially identical to any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, or 16; b) a polynucleotide including a sequence substantially identical to any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, or 15; c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) or (b) and encodes a Greatwall polypeptide; d) a polynucleotide having a sequence that deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a Greatwall polypeptide; and e) a polynucleotide which represents a fragment, derivative or
  • the invention features an expression vector containing any of the aforementioned polynucleotides; a host cell containing such expression vectors; and a Greatwall polypeptide encoded by any of these polynucleotides or expression vectors.
  • the invention features a method for producing a Greatwall polypeptide, wherein the method includes the following steps: a) culturing any of the aforementioned host cells under conditions suitable for the expression of the Greatwall polypeptide; and b) recovering the Greatwall polypeptide from the host cell culture.
  • the invention features a method for detection of a polynucleotide encoding a Greatwall polypeptide in a biological sample including the following steps: a) hybridizing any polynucleotide described herein to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, hi preferred embodiments, before hybridization, the nucleic acid material of the biological sample is amplified (e.g., by using a standard polymerase chain reaction).
  • the invention features a method for detection of a Greatwall polynucleotide or a Greatwall polypeptide, including the step of contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the Greatwall polypeptide. Diagnostic kits for performing these methods are further provided.
  • the invention features a method of screening for agents which decrease the activity of a Greatwall polypeptide, including the steps of: a) contacting a test compound with any Greatwall polypeptide encoded by any polynucleotide described herein; and b) detecting binding of the test compound to the Greatwall polypeptide, wherein a test compound which binds to the Greatwall polypeptide is identified as a potential therapeutic agent for decreasing the activity of a Greatwall polypeptide.
  • the invention further features a method of screening for agents which regulate the activity of a Greatwall polypeptide, including the steps of: a) contacting a test compound with a Greatwall polypeptide encoded by any polynucleotide described herein; and b) detecting Greatwall polypeptide activity, wherein a test compound which increases the Greatwall polypeptide activity is identified as a potential therapeutic agent for increasing the activity of the Greatwall polypeptide, and wherein a test compound which decreases the Greatwall polypeptide activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the Greatwall polypeptide.
  • a method of screening for agents which decrease the activity of a Greatwall polypeptide is also included in the invention. This method includes the steps of: a) contacting a test compound with any polynucleotide described herein; and b) detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of the Greatwall polypeptide.
  • the invention includes a method of reducing the activity of a
  • the invention features a compound that modulates the activity of a Greatwall polypeptide or a polynucleotide wherein the compound is identified by any of the aforementioned methods.
  • Pharmaceutical compositions that include (i) the expression vectors described herein or the reagents identified according the disclosed methods and (ii) a pharmaceutically acceptable carrier are further provided. Use of the expression vectors described herein or the compounds identified herein in the preparation of a medicament for modulating the activity of a Greatwall polypeptide in a cell (e.g., a neoplastic or cancerous cell) is further provided.
  • the invention features an isolated Greatwall polypeptide including a first polypeptide segment including an amino acid sequence having substantial identity to any one of the amino acid sequences shown in SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, or 16.
  • Exemplary polypeptides further including a second polypeptide segment include an amino acid sequence which is not a human Greatwall polypeptide amino acid sequence, wherein the second polypeptide segment is joined to the first polypeptide segment by means of a peptide bond.
  • the invention features an isolated Greatwall polypeptide including an amino acid sequence which differs from the amino acid sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, or 16 by between one and ten conservative amino acid substitutions and which has a Greatwall polypeptide activity; and an isolated and purified polypeptide including a first polypeptide segment which includes between 50 and 100 contiguous amino acids of a Greatwall polypeptide as shown in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, or 16.
  • the polypeptide further includes a second polypeptide segment which is not a human Greatwall polypeptide amino acid sequence, wherein the second polypeptide segment is joined to the first polypeptide segment by means of a peptide bond.
  • the invention provides a purified antibody which specifically binds to a Greatwall polypeptide.
  • exemplary antibodies include polyclonal antibodies, monoclonal antibodies, single-chain antibodies, as well as Fab, F(ab') 2 , or a Fv fragment.
  • the invention features an isolated polynucleotide which encodes a Greatwall polypeptide including the amino acid sequence shown in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, or 16.
  • Such polynucleotides include the nucleotide sequence shown in any one of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, or 15.
  • the invention features a cDNA molecule which encodes a Greatwall polypeptide that is substantially identical to the amino acid sequence shown in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, or 16.
  • the cDNA includes the nucleotide sequence shown in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, or 15.
  • a cDNA molecule which encodes a portion of a Greatwall polypeptide including the amino acid sequence shown in any one of SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, or 16 is also provided.
  • Such cDNA molecules typically include a contiguous nucleotide sequence selected from any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, or 15.
  • the invention features an isolated and purified single- stranded probe including between 25 and 100 contiguous nucleotides of a sequence encoding a Greatwall polypeptide or the complement thereof, wherein the Greatwall polypeptide includes the amino acid sequence shown in any one of SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, or 16.
  • the probe includes any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11 , 13, or 15 or a fragment thereof.
  • the invention features an isolated antisense oligonucleotide including a first sequence of between 25 and 150 contiguous nucleotides which is complementary to a second sequence of between 25 and 150 contiguous nucleotides found in a coding sequence for a Greatwall polypeptide which includes the amino acid sequence shown in any one of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, or 16.
  • the invention features a kit including a set of primers, wherein the set includes: a first primer including at least 8 contiguous nucleotides which is complementary to a contiguous sequence of nucleotides located at the 5' end of the coding strand of a double-stranded polynucleotide which encodes a Greatwall polypeptide as shown in any one of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, or 15; and a second primer including at least 8 contiguous nucleotides which is complementary to a contiguous sequence of nucleotides located at the 5' end of the non-coding strand of the polynucleotide.
  • the coding strand includes the nucleotide sequence shown in any one of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, or 15.
  • the invention features an expression construct including (i) a sequence encoding a Greatwall polypeptide including the amino acid sequence shown in SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, or 16; and (ii) a promoter located upstream from the sequence and which controls expression of sequence.
  • the coding sequence includes the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, or 15.
  • the invention features a host cell including an expression construct, wherein the expression construct includes (i) a sequence encoding a Greatwall polypeptide including the amino acid sequence shown in any one of SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, or 16; and (ii) a promoter which is located upstream from the coding sequence and which controls expression of the coding sequence.
  • Exemplary host cells include expression vectors that contain a coding sequence having the nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, or 15.
  • Host cells may be prokaryotic or eukaryotic cells.
  • the invention features a method of producing a Greatwall polypeptide, including the steps of: (i) culturing a host cell in a culture medium, wherein the host cell includes an expression construct including (a) a sequence encoding a Greatwall polypeptide including the amino acid sequence as shown in any one of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, or 16; and (b) a promoter which is located upstream from the coding sequence and which controls expression of the coding sequence, wherein the step of culturing is carried out under conditions whereby the protein is expressed; and recovering the protein from the culture medium.
  • the invention features a method of detecting a Greatwall polypeptide expression product, including the steps of: (a) contacting a test sample with a reagent that specifically binds to an expression product of the Greatwall polypeptide coding sequence; (b) assaying the test sample to detect binding between the reagent and the expression product; and (c) identifying the test sample as containing a Greatwall polypeptide expression product if binding between the reagent and the expression product is detected.
  • the expression product is a polypeptide or an rnRNA molecule
  • the test sample is a cell or is a culture medium
  • the reagent is an antibody or an inhibitory nucleic acid.
  • the invention features a method of treating a Greatwall expression disorder, the method including the step of: administering to a patient with the disorder an effective amount of a reagent that either (a) decreases expression of a Greatwall polypeptide gene that encodes a Greatwall polypeptide having substantial identity to the amino acid sequence shown in any one of SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, or 16 or (b) decreases effective levels of the Greatwall polypeptide, whereby symptoms of the Greatwall expression disorder are reduced.
  • the reagent is an antibody that specifically binds to the Greatwall polypeptide or an inhibitory nucleic acid.
  • the invention features a method of treating a Greatwall expression disorder, the method including the step of: administering to a patient with cancer an effective amount of a Greatwall polypeptide agonist, a protein or an expression vector encoding a Greatwall polypeptide, whereby symptoms of the expression disorder are reduced, hi particular, the agonist is a small molecule.
  • the invention features a method of screening for candidate therapeutic agents that may be useful for treating cancer, the method including the steps of: (a) contacting a Greatwall polypeptide including an amino acid sequence having substantial identity to any one of the amino acid sequences shown in SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, or 16 with a test compound; assaying for binding between the Greatwall polypeptide and the test compound; and (b) identifying a test compound that binds to the Greatwall polypeptide as a candidate therapeutic agent that may be useful for treating cancer, hi preferred embodiments, the test compound or the Greatwall polypeptide includes a detectable label, hi other preferred embodiments, the test compound or the Greatwall polypeptide is bound to a solid support.
  • the invention features a method of screening for candidate therapeutic agents that may be useful for treating cancer including the steps of: (a) assaying for expression of a polynucleotide having substantial sequence identity to a polynucleotide encoding a Greatwall polypeptide including the amino acid sequence of any one of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, or 16 in the presence and absence of a test compound; and (b) identifying a test compound that increases or decreases the expression as a candidate therapeutic agent that may be useful for treating cancer.
  • the step of contacting is in a cell or is in a cell-free in vitro translation system.
  • the invention features a pharmaceutical composition including (i) a reagent which binds to an expression product having substantial identity to a Greatwall polypeptide gene which encodes a Greatwall polypeptide including any one of the amino acid sequences shown in SEQ E) NOS :2, 4, 6, 8, 10, 12, 14, or 16; and (ii) a pharmaceutically acceptable carrier.
  • the reagent is an antibody or an inhibitory nucleic acid.
  • the invention features a pharmaceutical composition including (i) polypeptide having substantial sequence identity to any one of the amino acid sequences shown in SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, or 16; and (ii) a pharmaceutically acceptable carrier; as well as a pharmaceutical composition including (i) a polynucleotide having substantial identify to a polynucleotide encoding a Greatwall polypeptide including the amino acid sequence shown in SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, or 16; and (ii) a pharmaceutically acceptable carrier.
  • Exemplary polynucleotide includes the nucleotide sequence shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, or 15.
  • the invention features a microarray including at least two Greatwall polynucleotides or fragments thereof; as well as a microarray including at least two Greatwall polypeptides or fragments thereof.
  • the invention features a method for identifying a compound that is capable of decreasing the expression or activity of a Greatwall polynucleotide, the method including (a) providing a Drosophila animal expressing a Greatwall polynucleotide; and (b) contacting the animal with a candidate compound, a decrease in Greatwall expression or activity following contact of the animal with the candidate compound identifying a compound that decreases the expression or activity of a Greatwall polynucleotide.
  • the invention features a method for identifying a compound for ameliorating or delaying an impaired mitotic condition, the method including the steps of: (a) providing a gwl mutant animal; and (b) contacting the gwl animal with a candidate compound, wherein progression through mitosis is an indication that the candidate compound ameliorates or delays an impaired mitotic condition.
  • the invention features a method of treating a Greatwall kinase dysfunction related disease, including the step of: administering to a patient in need thereof a therapeutically effective dose of a reagent that modulates a function of a Greatwall kinase, whereby symptoms of the Greatwall kinase dysfunction related disease are ameliorated.
  • Exemplary reagents are typically identified according to any of the methods described herein. The methods described herein are especially useful for treating diseases such as cancer.
  • isolated polynucleotide is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences, hi addition, the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • polypeptide is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
  • an “isolated polypeptide” is meant a Greatwall polypeptide that has been separated from components that naturally accompany it.
  • the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally- occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a Greatwall polypeptide.
  • An isolated Greatwall polypeptide may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method,, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 70% identity to a reference amino acid sequence (for example, any one of the amino acid sequences (e.g., SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16) described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences (e.g., SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15) described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences (e.g., SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16) described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences (e.g., SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15) described herein).
  • such a sequence is at least 75%, 80%, 85%, 90%, or 95%, more preferably 96%, and most preferably 97% or even 98% or 99% identical at the amino acid level or nucleic acid to
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e "3 and e "1 indicating a closely related sequence.
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology
  • transformed cell is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a polynucleotide molecule encoding (as used herein) a Greatwall polypeptide.
  • positioned for expression is meant that a Greatwall polynucleotide (e.g., a DNA molecule) is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant Greatwall polypeptide, or an RNA molecule).
  • a Greatwall polynucleotide e.g., a DNA molecule
  • a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant Greatwall polypeptide, or an RNA molecule).
  • purified antibody is meant an antibody which is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody.
  • a purified antibody of the invention may be obtained, for example, by affinity chromatography using a recombinantly-produced polypeptide of the invention and standard techniques.
  • telomere binding By “specifically binds” is meant a compound or antibody which recognizes and binds a Greatwall polypeptide but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a Greatwall polypeptide.
  • derived from is meant isolated from or having the sequence of a naturally-occurring sequence (e.g., a cDNA, genomic DNA, synthetic, or combination thereof).
  • immunological assay an assay that relies on an immunological reaction, for example, antibody binding to an antigen.
  • immunological assays include ELISAs, Western blots, immunoprecipitations, and other assays known to the skilled artisan.
  • inhibitory nucleic acid is meant a nucleic acid that reduces or eliminates expression or biological activity of a gene or protein of interest.
  • Inhibitory nucleic acids include, without limitation, antisense nucleic acids, double stranded RNAs (dsRNA), or small interfering RNAs (siRNA), or analogs thereof.
  • antisense is meant a nucleic acid, or analog thereof, regardless of length, that is complementary to the coding strand or mRNA of a nucleic acid sequence, hi one embodiment, an antisense RNA is introduced to an individual cell, tissue, organ, or to a whole animal. Desirably the antisense nucleic acid is capable of decreasing the expression or biological activity of a nucleic acid or amino acid sequence. In one embodiment, the decrease in expression or biological activity is at least 10%, relative to a control, more desirably 25%, and most desirably 50%, 60%, 70%, 80%, 90%, or more.
  • the antisense nucleic acid may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.
  • double stranded RNA is meant a complementary pair of sense and antisense RNAs regardless of length, hi one embodiment, these dsRNAs are introduced to an individual cell, tissue, organ, or to a whole animal. For example, they may be introduced systemically via the bloodstream. Desirably, the double stranded RNA is capable of decreasing the expression or biological activity of a nucleic acid or amino acid sequence, hi one embodiment, the decrease in expression or biological activity is at least 10%, relative to a control, more desirably 25%, and most desirably 50%, 60%, 70%, 80%, 90%, or more.
  • the antisense nucleic acid may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.
  • modified backbone for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.
  • dsRNAs may also be engineered to form in viro using standard methods known in the art.
  • siRNA is meant a dsRNA that complements a region of an mRNA.
  • an siRNA is 22-24 nucleotides in length and has a 2 base overhang at its 3' end.
  • These dsRNAs can be introduced to an individual cell or to a whole animal, for example, they may be introduced systemically via the bloodstream.
  • Such siRNAs are used to down-regulate mRNA levels or promoter activity.
  • the decrease in expression or biological activity is at least 10%, relative to a control, more desirably 25%, and most desirably 50%, 60%, 70%, 80%, 90%, or more.
  • the siRNA may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.
  • hybridize is meant to form a double-stranded molecule between complementary polynucleotide sequences (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, or 15) described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, or 15
  • stringent salt concentration will ordinarily be less than about 750 niM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37 0 C, and most preferably of at least about
  • hybridization time the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25°C, more preferably of at least about 42°C, and most preferably of at least about 68°C.
  • wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another preferred embodiment, wash steps will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1 % SDS . In a most preferred embodiment, wash steps will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. ScL, USA 72:3961, 1975); Ausubel et al. (Current
  • ortholog is meant a polypeptide or nucleic acid molecule of an organism that is highly related to a reference protein, or nucleic acid sequence, from another organism.
  • An ortholog is functionally related to the reference protein or nucleic acid sequence. In other words, the ortholog and its reference molecule would be expected to fulfill similar, if not equivalent, functional roles in their respective organisms.
  • a Drosophila Greatwall polypeptide and its mammalian ortholog would both be expected to fulfill its mitotic function of Greatwall in their respective organisms. It is not required that an ortholog, when aligned with a reference sequence, have a particular degree of amino acid sequence identity to the reference sequence.
  • a protein ortholog might share significant amino acid sequence identity over the entire length of the protein, for example, or, alternatively, might share significant amino acid sequence identity over only a single functionally important domain of the protein. Orthologs may be identified using methods provided herein. The functional role of an ortholog may be assayed using methods well known to the skilled artisan, and described herein.
  • function might be assayed in vivo or in vitro using a biochemical, immunological, or enzymatic assays; transformation rescue, assays for the effect of gene iiiactivation mitosis; such assays, as described herein, may be carried out in a whole animal (e.g., Drosophila) or in tissue culture; function may also be assayed by gene inactivation (e.g., by RNAi, siRNA, or gene knockout), or gene over-expression, as well as by other methods.
  • gene inactivation e.g., by RNAi, siRNA, or gene knockout
  • Greatwall polypeptide is meant a polypeptide that modulates mitotic progression of an organism.
  • a Greatwall polypeptide has substantial sequence identity to the proteins encoded by the nucleic acid sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 described herein.
  • Exemplary Greatwall polypeptides include SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and 16.
  • Greatwall polynucleotide is meant a nucleic acid that encodes a Greatwall polypeptide.
  • Exemplary polypeptides are encoded by the nucleic acid sequences nucleic acid sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 described herein.
  • Greatwall polypeptides are encoded by polynucleotides that have substantial identity to any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, or 15.
  • transgene any piece of DNA which is inserted by artifice into a cell and typically becomes part of the genome of the organism which develops from that cell.
  • a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
  • transgenic any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell.
  • the transgenic organisms are generally transgenic vertebrates, such as, zebrafish, mice, and rats, and the DNA (transgene) is inserted by artifice into the nuclear genome.
  • Cell as used herein may be a single-cellular organism, cell from a multi ⁇ cellular organism, or it may be a cell contained in a multi-cellular organism.
  • “Differentially expressed” means a difference in the expression level of a nucleic acid or a polypeptide. This difference may be either an increase or a decrease in expression, when compared to control conditions.
  • Microarray means a collection of polynucleotides or polypeptides from one or more organisms arranged on a solid support (for example, a chip, plate, or bead). These polynucleotides or polypeptides may be arranged in a grid where the location of each nucleic acid or polypeptide remains fixed to aid in identification of the individual polynucleotides or polypeptides.
  • a microarray may include, for example, polynucleotides representing all, or a subset, of the open reading frames of an organism, or of the polypeptides that those open reading frames encode.
  • the polynucleotides of the array are defined as having a common region of the genome having limited homology to other regions of an organism's genome.
  • a microarray may also be enriched for a particular type of gene.
  • a "microarray of Greatwall polynucleotides or polypeptides" maybe enriched for Greatwall polynucleotides or polypeptides so that, for example, it comprises at least 5%, 10%, 15%, 20%, 22%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97% or even 99% Greatwall genes or their encoded Greatwall polypeptides.
  • a "microarray of Greatwall polynucleotides or polypeptides” comprises the polynucleotides described herein, or the polypeptides they encode.
  • "Primer set” means a set of oligonucleotides that may be used, for example, for PCR.
  • a primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.
  • Therapeutic agent or "therapeutic compound” means a substance that has the potential of affecting the function of an organism.
  • Such an agent or a compound may be, for example, a naturally occurring, semi-synthetic, or synthetic agent.
  • the test compound maybe a drug that targets a specific function of an organism.
  • a test agent or compound may also be an antibiotic or a nutrient.
  • a therapeutic compound may decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease or disorder in a eukaryotic host organism.
  • the invention provides a number of targets that are useful for the development of drugs to treat neoplasia (such as cancer) and the dysregulation of mitosis.
  • the methods of the invention provide a facile means to identify therapies that are safe for use in eukaryotic host organisms (i.e., compounds which do not adversely affect the normal development, physiology, or fertility of the organism).
  • the methods of the invention provide a route for analyzing virtually any number of compounds for effects on mitosis with high- volume throughput, high sensitivity, and low complexity. The methods are also relatively inexpensive to perform and enable the analysis of small quantities of active substances found in either purified or crude extract form.
  • Figure 1 shows chromosome condensation defects in gwl mutant brain cells. Wild-type (A and D), gwl 716 mutant (B and E), and gwl 2970 matant (C and F) brains were treated with colchicine and the chromosomes were stained with either orcein (A-
  • Figure 2 shows chromosomes from gwl mutants contain the condensin component Barren and phospholiistone H3.
  • D and H Merge of signals for DlS[A (blue), phosphohistone H3 (red), and
  • Figure 3 shows the mitotic phenotype of gwl mutants. Wild-type (A-D) and gwl 716 mutant (E-H) neuroblasts stained for phosphohistone H3 (green) and tubulin (red); overlap is yellow.
  • a and E Prophase/prometaphase; note the undercondensation in gwl mutant chromosomes at this stage.
  • B and F metaphase; some chromosomes in gwl metaphases often fail to progress to the metaphase plate.
  • FIG. 5 shows division of colchicine-treated cultured neuroblasts in real time.
  • A Wild type; time in h and min (h:min).
  • B gwl 716 mutant neuroblast; time in h, min, and s (h:min:s).
  • First and third rows histone H2Av-GFP signal; second and fourth rows, DIC of the same cell at the same time.
  • Arrows are provided to track individual cells over time. Both wild-type and mutant cells arrest in metaphase, but the gwl mutant cells require much more time to condense their chromosomes prior to NEB. Bar, 5 ⁇ m.
  • Figure 6 shows DNA synthesis in gwl mutant brains. BrdU incorporation in wild-type (A) and gwl 2970 mutant brains (B and C). Bar, 100 ⁇ m.
  • Figure 7 shows that the spindle checkpoint is active in metaphase-arrested gwl mutant cells.
  • A-H Cyclin B levels are elevated in gwl metaphase cells.
  • A-D Wild type, with metaphase cell (high cyclin B) at bottom and anaphase/telophase cells (low cyclin B) at top;
  • E-H gwl 2970 mutant metaphase cell.
  • a and E DNA;
  • B and F cyclin B;
  • C and G phosphohistone H3;
  • D and H merge with DNA (blue), phosphohistone H3 (red), and cyclin B (green).
  • I-T Bubl is kinetochore-associated in gwl metaphase cells.
  • (L, P, and T) merge with DNA (blue), phosphohistone H3 (red), and Bubl (green). Bar, 5 ⁇ m.
  • Figure 8 shows that CG7719 is the gwl gene. The gwl region in interval 91 C.
  • Mutant gwl alleles fail to complement the deletions Df(3R)ChaM 5 and Df(3R)Cha9, but do complement Df(3R)Dl-FX2.
  • Thin lines show sequences removed by the corresponding deletions
  • thick lines are sequences not removed by the deletions
  • gray areas denote the region of uncertainty in which the breakpoints defining the position of gwl can lie.
  • the genes in the gwl region and their direction of transcription are shown. Left to right arrow corresponds to the centromere-to- telomere direction for chromosome arm 3R.
  • Figure 9 shows depletion of Greatwall by KNfA interference.
  • RNAi with g-w/ dsRNA RNAi with g-w/ dsRNA.
  • E RNAi with ds " RNA for Gluon (XCAP-C).
  • F with dsRNA for Barren (XCAP-H).
  • Figure 10 shows the characterization of an anti-Greatwall antibody.
  • A Western blot showing the absence or reduction of the CG7719 gene product isoforms in various gwl alleles. Extracts from 10 dissected larval brains of the indicated genotypes were loaded on each lane; the loading control (bottom) is a weakly cross- reacting band observed after prolonged exposure of the blot.
  • B The Greatwall protein is depleted from Kc tissue culture cells treated with gwl dsRNA. Control is extract from untreated cells.
  • Figure 11 shows that chromosomes contain topoisomerase II in cells deficient for Greatwall and Cohesin components.
  • Control or dsRNA-treated tissue culture cells were analyzed for tubulin, DNA, and topoisomerase II staining as shown.
  • the merged figures in the right column show tubulin in green, DNA in blue, and topoisomerase II in red. Colocalization of DNA and topoisomerase II (purple) is observed in cells depleted of Greatwall, Barrren, and Gluon, as well as in control cells. Bar, 5 ⁇ m.
  • Figure 12 shows the structure of Greatwall proteins. conserveed domains of the D. melanogaster and human Greatwall proteins, compared with the related kinases Riml5p/Ceklp and IRE. The kinase domain in all these proteins is split by the insertion of unrelated amino acids between kinase subdomains VII and VIII. Green and blue areas are less well-conserved regions found in all Greatwall kinases but not in other kinases (excepting the short green stretch preceding the kinase domains in Riml5p/Ceklp and IRE). A small degree of homology is apparent between the inserts in insect and vertebrate Greatwall proteins (yellow).
  • Figure 13 shows the Greatwall protein localizes to nuclei.
  • A-H Wild-type brains
  • I-L gwl 2970 mutant brain showing background levels of staining.
  • Figure 14 shows that Greatwall is a mitotic phosphoprotein with kinase activity.
  • A Greatwall is hyperphosphorylated during mitosis. Left panel: Endogenous Greatwall from CSF and interphase Xenopus extracts was detected by immunoblotting. Right panel: Greatwall from either CSF or interphase extracts was immunoprecipitated, treated with lamada phosphatase (PPS), and detected by Western blot. After phosphate treatment, CSF Greatwall migrated at the same position as interphase Greatwall.
  • PPS lamada phosphatase
  • Figure 15 shows that such extracts regain a mitotic-like state independent of Greatwall.
  • the translated Greatwall proteins were purified on anti-Greatwall beads prior to incubation with [7 "32 P]ATP. Wild-type Greatwall incorporated >10X more radioactivity than either kinase-dead form.
  • the control lane is an immunoprecipitate from extract without exogenous mRNA; an aliquot of M-GwI from part B was also run for comparison. Because of unequal efficiency of transcription/translation in this experiment, protein amounts were determined by Western blot (not shown) and then normalized prior to the kinase assay.
  • Figure 16 shows that Greatwall kinase is required to maintain MPF activity in
  • MAP kinase and MEK remain in their phosphorylated, active states for at least 10 min post Greatwall depletion, indicating that the rapid exit from M phase is not mediated by the MAP kinase pathway.
  • Figure 17 shows the rescue of Greatwall depletion.
  • Myc-tagged wildtype or kinase dead (G41S, D173A) Greatwall translated in CSF extracts was precipitated on anti-Myc beads, which were then added to Greatwall-depleted CSF extracts. Extracts reenter M phase within 5-10 min when supplemented with beads containing wildtype Greatwall, but not with control beads (isolated from CSF extracts without exogenous rnRNA) or beads containing mutant kinase-dead forms of Greatwall.
  • G41S and D 173 A Greatwall are rapidly dephosphorylated in extracts that remain in interphase.
  • Figure 18 shows the depletion of Greatwall kinase prevents mitotic entry.
  • A Cycling extracts were either mock-depleted with protein A beads or immunodepleted with anti-Greatwall beads. The control extract entered mitosis by 30 min and then rapidly exited M phase, while Greatwall-depleted extracts never entered mitosis.
  • Myc-tagged wildtype or kinase dead mutant (G41S; D 173 A gave identical results not shown) were translated from exogenous mRNAs added to interphase (I) or CSF (M) extracts. The tagged proteins were purified on anti-Myc beads that were then added to Greatwall-depleted cycling extracts.
  • Figure 20 shows that phosphorylations by MPF, but not Plxl, activate Greatwall's kinase activity, as measured by autophosphorylation and kinase assays using MBP (myelin basic protein) as substrate.
  • Figure 21 shows that the appearance of Hl kinase activity during cell cycle without Greatwall kinase is mainly due to Cdc2/cyclin A, but not Cdc2/cyclin B (which is MPF).
  • A CSF extracts.
  • B cycling extracts.
  • Figure 22 shows that, in cycling extracts, Greatwall depletion does not activate caffeine sensitive checkpoint.
  • A shows that caffeine can override G2/M checkpoint activated by DNA damage.
  • B shows that caffeine cannot override the slow mitotic entry after Greatwall depletion.
  • Figure 23 shows that the Greatwall depletion effect on CSF extracts can be partially rescued by adding exogenous PIx 1 and Cdc2-AF, meaning at certain signal transduction pathways, Plxl and Cdc2 are downstream of Greatwall kinase.
  • Chromosomes are highly undercondensed, particularly in the euchromatin, but nevertheless contain phosphorylated histone H3, condensin, and topoisomerase II. Cells take much longer to transit the period of chromosome condensation from late G 2 through nuclear envelope breakdown. Mutant cells are also subsequently delayed at metaphase, due to spindle checkpoint activity. These mutant phenotypes are caused neither by spindle aberrations, by global defects in chromosome replication, nor by activation of a caffeine-sensitive checkpoint.
  • the Greatwall proteins in insects and vertebrates are located in the nucleus and belong to the AGC family of serine/threonine protein kinases; the kinase domain of Greatwall is interrupted by a long stretch of unrelated amino acids.
  • Neuroblasts in gwl mutants have undercondensed chromosomes that nonetheless bind phosphohistone H3 and condensin
  • chromosome undercondensation The most obvious phenotype associated with gwl mutations is chromosome undercondensation. This is apparent in mutant larval brains whose chromosomes were stained with either orcein or Hoechst 33258 (Fig. 1), and is most visible in brains treated with colchicine, which arrests cells under conditions that promote chromosome condensation. In these chromosomes, the euchromatin is highly undercondensed, while the heterochromatin remains more compacted. Many aberrantly condensed chromosomes were nonetheless clearly composed of two sister chromatids.
  • gwl mutant chromosomes still reacted with antibodies directed against phosphohistone H3 and the condensin component Barren (the fly homolog of XCAP-H; Fig. 2).
  • the signal intensities on gwl chromosomes were roughly similar to those seen in wild-type, but Barren and phosphohistone H3 were more diffuse on the undercondensed mutant chromosomes.
  • gwl mutants Cytological examination of fixed brains from larvae homozygous or hemizygous for all five gwl alleles revealed defective mitotic progression.
  • the mitotic index was ⁇ 2.5 X higher than controls, and the proportion of mitotic cells that were in anaphase or telophase was only about 10-15% of that in wild-type (Table 1).
  • gwl mutant cells delay or arrest prior to anaphase onset. Almost all of the few residual ana/telophases in gwl brains were aberrant, with lagging chromosomes or chromosome bridges.
  • the spindles in gwl mutant cells were nevertheless morphologically normal at all mitotic stages (see Fig. 3).
  • telophase percentage total anaphase + telophase figures/total mitotic figures X 100.
  • a More than 600 mitotic figures were examined for each genotype. Percentage was defined as the number of mitotic cells at the specified stage/total number of mitotic cells X 100. Stages were determined by staining with anti-phosphohistone H3 and anti-lamin as described in the Materials and methods (infra).
  • mutant phenotype An alternative explanation for the mutant phenotype is that absence of Greatwall might activate a G 2 /early prophase checkpoint that reverses the early stages of chromosome condensation and delays NEB (Pines and Rieder, Nat. Cell Biol. 3. ⁇ 3-E6, 2001; Mikhailov and Rieder, Cur. Biol. 12.-R331-R333, 2002). Many checkpoints of this type monitor DNA damage and are mediated by the ATM and ATR kinases; when activated, these checkpoints inhibit cyclin B-CDK via the inhibition of Cdc25 phosphatase (reviewed by Abraham, Genes Dev. 15:2177-2196, 2001).
  • the checkpoint protein Bubl accumulates on gwl mutant kinetochores, as is true in wild-type cells prior to anaphase onset (Fig. 7 I-T). That the metaphase arrest in gwl mutants is due to the spindle checkpoint is consistent with published reports showing that improper chromosome condensation or DNA damage can disrupt the centromere/kinetochore and delay satisfaction of the checkpoint (Pflumm and Botchan, Development 128:1697-1707, 2001; Garber and Rine, Genetics 161:521- 534, 2002; Mikhailov et al., Cur. Biol. 12:1797-1806, 2002).
  • RNA interference RNA interference
  • dsRNA Chromosomes from cells treated with CG7719 double-stranded RNA (dsRNA) were highly undercondensed (Fig. 9), reminiscent of the chromosomes in gwl mutant brains. This undercondensation is strikingly similar to that observed when fly tissue culture cells were treated with dsRNA for two condensin complex components (Gluon [XCAP-C] and Barren [XCAP-H]; Fig. 4 E-H). Greatwall- depleted cells also showed a 2-3X increase in the mitotic index and a 3X decrease in the percentage of ana/telophase cells (data not shown), consistent with the mitotic delays in gwl brains.
  • Greatwall belongs to the AGC family, a diverse group of serine/threonine kinases that phosphorylate targets surrounded by basic amino acids (Hanks and Hunter, FASEB J. 9:576-596, 1995; Morrison et al., J. Cell Biol. 150:F57-F62, 2000).
  • the kinase domain of Greatwall is split (Fig. 12), with -500 amino acids separating subdomains VII and VIII (Hanks and Quinn, Methods Enzymol. 200:38-62, 1991).
  • Other insects and vertebrates including humans have proteins very closely related to Greatwall; the kinase domains of the fly and human Greatwall proteins share 59% overall amino acid sequence identity (Fig. 12).
  • the homologies between Greatwall proteins extend beyond the kinase domain into the flanking blue and green regions shown on Fig. 12.
  • the insertions of several hundred amino acids between subdomains VII and VIII are less conserved, but we can still detect limited homology between the insect and vertebrate insertions.
  • the Greatwall proteins all form a single homology group: they are the closest relatives found in all pairwise searches between these species.
  • Greatwall is more distantly related to several other proteins that also contain an interruption between kinase subdomains VII and VIII, including ERE and IREHl in Arabidopsis, CEKl in S. pombe, and RIM15 in S. cerevisiae.
  • the Greatwall protein localizes to the nucleus
  • We used anti-Greatwall antibodies as immunofluorescence probes see Fig. 10 for the characterization of these antibodies.
  • Greatwall accumulated in a punctate pattern in the nuclei of all interphase and prophase cells (Fig. 13). The distribution of the protein within nuclei does not match that of the DNA, but some Greatwall protein might still be chromosome-associated.
  • Greatwall was relatively evenly distributed throughout mitotic cells, with no obvious accumulation over the chromosomes or the spindle. The nuclear localization of Greatwall was verified by overexpressing GFP-tagged Greatwall in tissue culture cells (data not shown).
  • chromosome undercondensation in gwl mutants reflects neither a lack of histone H3 phosphorylation, nor a failure of condensin or topoisomerase II to associate with chromosomes (Figs. 2 and 10).
  • Df(3R)Cha9, Df(3R)Dl-FX2 and Df(3R)ChaM5 were from the Bloomington Drosophila stock center. Oregon-R was the wild-type strain. Mutations and deficiencies were balanced over TM6B with Humeral (Hu) and Tubby
  • Tb over TM6C with Stubble (Sb) and Tb.
  • Sb Stubble
  • gwlll ⁇ was crossed with a stock containing a GFP-tagged histone H2AvD transgene on the third chromosome (Clarkson and Saint, DNA Cell Biol. 18:457-462, 1999). Desired recombinants were rebalanced over TM6C.
  • Mouse anti-lamin antibody was the gift of Dr. Paul Fisher (SUNY Stony Brook, Stony Brook, NY; Stuurman et al., J. Cell Sci. 108:3137-3144, 1995), and rabbit anti-Barren was from Dr. Hugo Bellen (Baylor College of Medicine, Houston, TX; Bhat et al., Cell 87:1103-1114, 1996).
  • sequences from a gwl cDNA clone (LD35132) encoding amino acids 371- 846 were inserted into the pMAL-C2 vector (New England Biolabs, Beverly, MA).
  • Greatwall is a mitotic phosphoprotein capable of autophosphorylation Antibodies prepared against Xenopus Greatwall epitopes recognize a single band of the expected molecular weight (98 kDal) in interphase extracts (Fig. 14A).
  • CSF mitotic
  • Greatwall's electrophoretic mobility is retarded. This mobility shift is mostly due to phosphorylation, since phosphatase-treated CSF Greatwall migrates identically with interphase Greatwall (Fig. 14A).
  • Greatwall is phosphorylated at multiple sites, because several bands of intermediate mobility appear soon after CSF extracts are induced to exit mitosis by calcium addition (see Fig. 16B below).
  • Xenopus Greatwall indeed contains one strong consensus MPF phosphorylation site (S/TPXK/R), as well as 11 less stringent sites (S/TP); some of these are near an RXL motif which helps inhibitors, activators, and substrates of cyclin dependent kinases bind to cyclin subunits (reviewed by Wohlschlegel et al., MoI Cell Biol. 21 :4868-74, 2001). Verification that Greatwall is an MPF substrate must nonetheless await demonstration that the sites on Greatwall phosphorylated by MPF in vitro are also targeted in vivo.
  • Fig. 16B demonstrate that Greatwall depletion does not immediately affect the MAPK pathway.
  • MAPK remained in its active, phosphorylated form for at least 10 minutes after MPF was inactivated and Mytl and Cdc25 were dephosphorylated. The same was true for MEK, MAPK's upstream activator (Fig. 16B), and for ⁇ 90 ⁇ , MAPK's downstream target that inactivates Mytl and Weel (control in Fig. 17; see Tunquist and Mailer, Genes Dev. 17:683-710,
  • Greatwall might be a direct substrate of MPF, but Greatwall might instead be indirectly activated by downstream mitotic kinases.
  • Greatwall's kinase activity is clearly required for its cell cycle functions, as kinase-dead enzyme does not rescue the effects of Greatwall depletion in CSF or cycling extracts.
  • Greatwall autophosphorylation probably provides one of the steps involved in its full activation, but verification of this point must await identification of the specific sites targeted by autophosphorylation and by upstream kinases.
  • PIx 1 and Cdc2 co-immunoprecipitates with Greatwall kinase PIx 1 was found to associate with Greatwall kinase in both M-phase and interphase, while Cdc2 kinase co-immunoprecipitates with Greatwall kinase during M-phase (Fig. 19).
  • Greatwall protein was expressed and purified from insect cells using a standard Bac- to-Bac system (Invitrogen, Carlsbad, CA). After a 60 min incubation in either CSF extracts or interphase extracts, Greatwall protein was co-immunoprecipitated from the extracts using anti-6His antibody (Cell Signaling Inc., Beverly, MA).
  • the phosphorylation sites of Greatwall were determined as follows. Unphosphorylated Xenopus Greatwall protein was expressed in and purified from insect cells using Bac-to-Bac system (Invitrogen, Carlsbad, CA). Next, Greatwall protein and active MPF were incubated in a kinase reaction at 30°C for 2 hours. The kinase reaction was stopped by addition of 2X Laemmli Sample Buffer (Bio-Rad, Hercules, CA), boiled for 3 min, and resolved on SDS-PAGE gel. The Greatwall band was visualized by Coomassie blue stain and digested by trypsin. After trypsin digestion, the resulting peptides were extracted from gel pieces. Phosphopeptides were identified by LC/MS/MS Mass Spectrometry according to standard methods. This analysis identified 12 MPF phosphorylation sites on Greatwall kinase as listed in Table 4 (below).
  • Hl kinase activity during cell cycle without Greatwall kinase is mainly due to Cdc2/cyclin A, but not Cdc2/cyclin B (which is MPF).
  • Greatwall depletion was performed in either CSF or cycling extracts according to standard methods. At designated time points, extracts were immediately frozen in liquid nitrogen. One set of these samples was resolved on SDS-PAGE gel followed by Western blot analysis of Greatwall, Cyclin A, Cyclin B, and Phospho- Tyrl5 of Cdc2. The second sample set was assayed for Hl kinase activity, using total extracts, or Cyclin A immunoprecipitates, or Cyclin B immunoprecipitates.
  • Plxl and Cdc2-AF were expressed and purified from insect cells using Bac-to- Bac system (Invitrogen, Carlsbad, CA). The purified proteins were next added into CSF extracts and then Greatwall depletion was performed according to the methods described herein. As is shown in Figure 23, the Greatwall depletion effect on CSF extracts was partially rescued by adding exogenous Plxl and Cdc2 ⁇ AF, indicating that Plxl and Cdc2 signal transduction pathways are downstream of Greatwall kinase.
  • Cdc2 monoclonal antibody (sc-54), and Mos antibody (sc-86) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
  • Antibodies against Phospho- MAPK (#9106), Phospho-MEK (#9121), Phospho-Cdc2 (Tyrl5) (#9111), and c-Myc (#9402) were from Cell Signaling Technology (Beverly, MA).
  • Plxl monoclonal antibody and anti-p90 Rsk were from Zymed (South San Francisco, CA).
  • Mytl and Cdc25 antibodies were the gifts of Dr.
  • Cyclin B2 antibody was provided by Dr. James Mailer (University of Colorado, Denver, CO).
  • Antibody to Xenopus Weel was generated by cloning a full length Weel cDNA into pQE30 (Qiagen, Hilden,
  • This construct made a 6His-Weel fusion protein in E. coli. Serum from immunized rabbits was affinity-purified on immobilized 6His-Weel. Xenopus EgR Extracts Cycling and CSF extracts, as well as demembranated frog sperm nuclei were prepared as described (Murray, Methods Cell Biol. 36:581-605, 1991). Interphase extracts were made by adding CaCl 2 to CSF extracts (0.5 mM final concentration); the extracts were then incubated at 23 0 C for 40 min to induce mitotic exit. Immunodepletion was performed as described (Sharp-Baker and Chen, J Cell Biol.
  • the plasmids were linearized with Seal, and then used as templates to produce mRNAs using the mMESSAGE mMACHINE Transcription Kit (Ambion, Austin, TX).
  • mMESSAGE mMACHINE Transcription Kit Ambion, Austin, TX.
  • the pMAL-GWL fusion protein To make bacterially synthesized Greatwall, the pMAL-GWL fusion protein
  • Immunoprecipitates were washed 3X in extract buffer (XB: 10 mM Hepes, pH 7.8, 50 mM sucrose, 100 mM KCl, 10 mM MgC12, 1 mM CaC12, 5 mM EGTA, and 10 ⁇ g/ml each of leupeptin, pepstatin, and chymostatin); and 2X in kinase buffer (20 mM Hepes, 10 mM MgC12, 0.1 mg/ml BSA, 3 mM /3-mercaptoethanol).
  • extract buffer XB: 10 mM Hepes, pH 7.8, 50 mM sucrose, 100 mM KCl, 10 mM MgC12, 1 mM CaC12, 5 mM EGTA, and 10 ⁇ g/ml each of leupeptin, pepstatin, and chymostatin
  • 2X in kinase buffer (20 mM Hepes, 10 mM M
  • the immunoprecipitates were resuspended in 10 ⁇ l of kinase buffer with 100 ⁇ M ATP and 10 ⁇ Ci [Y 32 P]ATP, and incubated at 30 0 C for 15 min. 1 ⁇ l of purified MPF (New England Biolabs) was added to test Greatwall phosphorylation by MPF. Reactions were terminated by addition of SDS-PAGE sample buffer, and boiled for 3 min before analysis by gel electrophoresis and autoradiography.
  • LPP Lambda Protein Phosphatase
  • the isolation of additional Greatwall nucleic acid sequences is made possible using the sequence described herein and standard techniques.
  • mammalian Greatwall sequences such as a human Greatwall sequence
  • invertebrate Greatwall sequences is made possible using the sequence described herein and standard techniques.
  • Greatwall oligonucleotide probes including degenerate oligonucleotide probes (i.e., a mixture of all possible coding sequences for a given amino acid sequence). These oligonucleotides may be based upon the sequence of either strand of the DNA.
  • Exemplary probes or primers for isolating mammalian Greatwall sequences preferably correspond to conserved blocks of amino acids as described herein.
  • General methods for designing and preparing such probes are provided, for example, in Ausubel et al., Current Protocols in Molecular Biology, 2003, Wiley & Sons, New York, N. Y.; and Guide to Molecular Cloning Techniques, 1987, S. L. Berger and A. R. Kimmel, eds., Academic Press, New York.
  • These oligonucleotides are useful for Greatwall gene isolation, either through their use as probes for hybridizing to Greatwall complementary sequences or as primers for various polymerase chain reaction (PCR) cloning strategies.
  • PCR polymerase chain reaction
  • the primers are optionally designed to allow cloning of the amplified product into a suitable vector.
  • oligonucleotide probes may be used for the screening of the recombinant DNA library.
  • the oligonucleotides are, for example, labelled with 32P using methods known in the art, and the detectably-labelled oligonucleotides are used to probe filter replicas from a recombinant DNA library.
  • Recombinant DNA libraries may be prepared according to ' methods well known in the art, for example, as described in Ausubel et al., supra, or may be obtained from commercial sources.
  • high stringency hybridization conditions may be employed; such conditions include hybridization at about 42 0 C and about 50% formamide; a first wash at about 65 0 C, about 2X SSC, and 1% SDS; followed by a second wash at about 65°C and about 0.1% SDS, IX SSC.
  • Lower stringency conditions for detecting Greatwall genes having less sequence identity to the Greatwall gene described herein include, for example, hybridization at about 42 0 C in the absence of formamide; a first wash at about 42 0 C, about 6X SSC, and about 1% SDS; and a second wash at about 5O 0 C, about 6X SSC, and about 1% SDS.
  • Greatwall oligonucleotides may also be used as primers in PCR cloning strategies.
  • Such PCR methods are well known in the art and described, for example, in PCR Technology, H. A. Erlich, ed., Stockton Press, London, 1989; PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H.
  • Greatwall polypeptides according to the invention may be produced by transformation of a suitable host cell with all or part of a Greatwall- encoding cDNA fragment (e.g., one of the cDNAs described above) in a suitable expression vehicle.
  • a suitable host cell with all or part of a Greatwall- encoding cDNA fragment (e.g., one of the cDNAs described above) in a suitable expression vehicle.
  • the Greatwall polypeptide may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, a plant cell, insect cells, e.g., Sf9 or Sf21 cells, or mammalian cells, e.g., COS 1, NIH 3T3, or HeLa cells).
  • a prokaryotic host e.g., E. coli
  • a eukaryotic host e.g., Saccharomyces cerevisiae, a plant cell, insect cells, e.g., Sf9 or Sf21 cells, or mammalian cells, e.g., COS 1, NIH 3T3, or HeLa cells.
  • Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., supra).
  • the method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles maybe chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).
  • baculovirus system using, for example, Sf9 cells and the method of Ausubel et al., supra.
  • Another baculovirus system makes use of the vector pBacPAK9 and is available from Clontech (Palo Alto, Calif.).
  • a Greatwall polypeptide is produced in a mammalian system, for example by a stably-transfected mammalian cell line.
  • a number of vectors suitable for stable transfection of mammalian cells are available to the public, e.g., see Pouwels et al. (supra); methods for constructing such cell lines are also publicly available, e.g., in Ausubel et al. (supra).
  • cDNA encoding the Greatwall protein is cloned into an expression vector which includes the dihydrofolate reductase (DHFR) gene.
  • DHFR dihydrofolate reductase
  • the Greatwall protein-encoding gene into the host cell chromosome is selected for by inclusion of 0.01-300 ⁇ M methotrexate in the cell culture medium (as described in Ausubel et al., supra). This dominant selection may be accomplished in most cell types. Recombinant protein expression may be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described in Ausubel et al. (supra); such methods generally involve extended culture in medium containing gradually increasing levels of methotrexate.
  • DHFR- containing expression vectors commonly used for this purpose include pCVSEII- DHFR and pAdD26SV(A) (described in Ausubel et al., supra).
  • Any of the host cells described above or, preferably, a DHFR-deficient CHO cell line e.g., CHO DHFR- cells, ATCC Accession No. CRL 9096
  • a DHFR-deficient CHO cell line are among the host cells preferred for DHFR selection of a stably-transfected cell line or DHFR-mediated gene amplification.
  • the Greatwall polypeptide is produced in vivo or, preferably, in vitro using a T7 system (see, for example, Ausubel et al., supra, or other standard techniques).
  • the recombinant Greatwall protein is expressed, it is isolated, e.g., using affinity chromatography.
  • an anti-Greatwall protein antibody e.g., produced as described herein
  • Lysis and fractionation of Greatwall protein-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra).
  • the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, eds., Work and Burdon, Elsevier, 1980).
  • Polypeptides of the invention particularly short Greatwall polypeptide fragments, may also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co.,
  • anti-Greatwall antibodies are produced as follows.
  • a Greatwall CDNA fragment encoding amino acids for example, SEQ ID NOS:2, 4, 5, 6, 8, 10, 12, 14, or 16
  • the fusion protein is then purified on a glutathione column, also by standard techniques, and is used to immunize rabbits.
  • the antisera obtained is then itself purified on a GST-Greatwall affinity column by the method of Finney and Ruvkun (Cell 63 :895-905, 1990). This antisera is shown to specifically identify GST-Greatwall by Western blotting.
  • MAL-Greatwall fusion protein is utilized as described herein.
  • Antibodies against human and Drosophila Greatwall proteins have also been generated according to the standard methods disclosed herein using Xenopus Greatwall protein.
  • Short peptide sequences for generating antibodies include the following: Human Greatwall Protein from 30 to 51(SEQ ID NO: 17), from 80 to 103 (SEQ ID NO: 18), from 110 to 147 (SEQ ID NO:19), from 157 to 181 (SEQ ID NO:20), from 184 to 233 (SEQ ID NO:21), from 311 to 331 (SEQ ID NO:22), from 360 to 380 (SEQ ID NO:23), from 444 to 464 (SEQ ID NO:24), from 510 to 530 (SEQ ID NO:25), from 686 to 750 (SEQ ID NO:26), from 771 to 791 (SEQ ID NO:27), and from 856 to 878 (SEQ ID NO:28);
  • Polypeptides for antibody production may be produced by recombinant or peptide synthetic techniques (see, e.g., Solid Phase Peptide Synthesis, supra; Ausubel et al., supra).
  • the peptides may, if desired, be coupled to a carrier protein, such as KLH as described in Ausubel et al, supra.
  • KLH-peptide is mixed with Freund's adjuvant and injected into guinea pigs, rats, or preferably rabbits.
  • Antibodies may be purified by any method of peptide antigen affinity chromatography.
  • monoclonal antibodies may be prepared using a Greatwall polypeptide (or immunogenic fragment or analog) and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N. Y., 1981; Ausubel et al., supra).
  • polyclonal or monoclonal antibodies are tested for specific Greatwall recognition by Western blot or immunoprecipitation analysis (by the methods described in Ausubel et al., supra).
  • Antibodies which specifically recognize Greatwall are considered to be useful in the invention; such antibodies may be used, e.g., in an immunoassay to measure or monitor the level of Greatwall produced by a mammal or to screen for compounds which modulate Greatwall production.
  • Anti- Greatwall antibodies may also be used to identify cells that express the Greatwall gene.
  • Fragments of antibodies may also be produced according to standard methods. Pepsin digestion, for example, may be used to cleave the intact anti-Greatwall antibodies into antibody fragments as follows. A buffer exchange with 10OmM sodium citrate (pH 3.5) using NAPTM- 10 columns (Amersham Pharmacia Biotech) can be used. Pepsin digestion can also be done with an unrelated human antibody (for example, Chrompure IgM, Dianova, Hamburg, Germany) to obtain a suitable negative control. For each milligram of antibody, 5 ⁇ g pepsin (Sigma Aldrich, Taufmaschinen, Germany) is added, followed by incubation for 10-15 minutes in a 37 °C water bath.
  • the reaction is stopped by adding 1/10 volume of 3.0 M Tris (pH 8.8) followed by centrifuging at 10,000 g for 30 minutes.
  • the fragmented Greatwall antibody and the fragmented human control antibody can be dialyzed against PBS.
  • the success of pepsin digestion may be analyzed by SDS- PAGE and Western blotting under non-reducing conditions. After blotting, the intact antibody may show the characteristic bands corresponding to intact antibody, monomeric forms, and light chains. By SDS-PAGE, the intact antibody may be unable to migrate into the stacking gel. However, following 10-15 minutes of treatment with pepsin, intact antibodies are completely digested into monomeric, F(ab) 2 , Fab, and light chain fragments which may be identified by molecular weight.
  • the fragmented Greatwall antibody may be tested for binding to tissue on paraffin sections of human carcinomas and compared to the intact Greatwall antibody. Both antibody forms may possess similar binding patterns on tumor cells.
  • cloning of the complementary-determining regions (CDRs) of anti-Greatwall antibodies may be performed as follows.
  • Total RNA from hybridomas which secrete a Greatwall-specific monoclonal antibody can be prepared according to a standard extraction procedure, and DNA fragments encoding the variable regions of the heavy and light chains can be amplified from poly(A)+ RNA.
  • the PCR products are then cloned into a vector such as pCR4-TOPO, ⁇ CR2.1-TOPO, or pB ADTMo- TOPO (Invitrogen) according to the manufacturer's instructions.
  • the resulting clones are amplified in E. coli TOPlO cells (Invitrogen) with ampicillin (Roche) as a selective marker.
  • Plasmid DNA is isolated from amplified clones using QIAGEN maxiprep kits, and nucleic acid sequencing is performed according to standard methods. Predicted amino acid sequences are then derived from the DNA sequences using Vector NTI (hiformax). On the basis of determining the predicted amino acid sequences, and according to the Chothia CDR definitions (Chothia et al., Nature, 342: 877 - 83, 1989), CDRs of each variable region of mouse monoclonal antibodies to Greatwall can be determined.
  • a patient suspected of having a neoplasm may be given a dose of radioiodinated Greatwall antibody and radiolabeled unspecific antibody.
  • An infusion of equal volumes of solutions of 131 I-Greatwall antibody and Tc-99m-labeled unspecific antibody are administered to a patient intravenously.
  • the patient Prior to administration of the reagents, the patient is typically pre-tested for hypersensitivity to the antibody preparation (unlabeled) or to antibody of the same species as the antibody preparation.
  • Lugol's solution is administered orally, beginning one or more days before injection of the radioiodinated antibody, at a dose of 5 drops twice or three-times daily.
  • Images of various body regions and views may be taken at 4, 8, and 24 hours after injection of the labeled preparations.
  • the neoplasm e.g., a carcinoma
  • the neoplasm is detected by gamma camera imaging with subtraction of the Tc-99m counts from those of 131 I, as described for 131 I -labeled anti-CEA antibody and Tc- 99m-labeled human serum albumin by DeLand et al. (Cancer Res. 40:3046, 1980).
  • imaging is usually clear and improves with time up to the 24 hour scans. Testing of Antibody-Based and Inhibitory Nucleic Acid Therapeutics in Animal Models (Xenografts) For various cancers
  • a nude mouse-human colon carcinoma cell system may be used.
  • Cells derived from a carcinoma cell line may be injected intraperitoneal (i.p.) into NMRI nu/nu mice.
  • i.p. intraperitoneal
  • Control mice receive the same quantity of unrelated fragmented human antibody (or inhibitory nucleic acids).
  • tumor growth can be controlled macroscopically. The mice may be sacrificed after a period of time that allows for tumor formation, for example 23 days.
  • mice may develop measurable tumors i.p. that increase in size over time.
  • mice treated with intact or fragmented Greatwall antibody (or inhibitory nucleic acids) may not develop detectable tumors, or may possess tumors that show a significant reduction in growth and size as compared to control mice.
  • mice injected with human carcinoma cells may also be conducted.
  • Mice inoculated i.p. with carcinoma cells may receive an intact or fragmented Greatwall antibody or a human control antibody (or inhibitory nucleic acids).
  • the control group may show expanded tumor spreading into the peritoneum, diaphragm, kidney, stomach, intestine, liver and spleen, whereas mice treated with the Greatwall antibody show a reduced spread of tumors to other organs and tissues.
  • the overall weight of tumor mass may also be reduced significantly by the Greatwall antibody treatment.
  • Morphological analysis of the tumors may reveal that tumors from mice treated with the Greatwall antibody exhibit not only reduced size, but also regressive changes in growth patterns like tumor-regression, infiltration ,and a high number of pyknotic cells.
  • In situ staining of the tumors for apoptotic activity may show that the tumors from mice treated with the Greatwall antibody have a significantly higher number of tumor cells undergoing apoptosis compared to control mice.
  • a patient diagnosed with a neoplasm may be treated with a Greatwall antibody or fragment thereof as follows.
  • Lugol's solution may be administered, e.g., 7 drops 3 times daily, to the patient.
  • a therapeutic dose of an 131 I-Greatwall antibody may be administered to the patient.
  • a 131 I dose of 50 mCi may be given weekly for 3 weeks, and then repeated at intervals adjusted on an individual basis, e.g., every three months, until hematological toxicity interrupts the therapy.
  • the exact treatment regimen is generally determined by the attending physician or person supervising the treatment.
  • the radioiodinated antibodies may be administered as slow intravenous infusions in 50 ml of sterile physiological saline. After the third injection dose, a reduction in the size of the primary tumor and metastases may be noted, particularly after the second therapy cycle, or 10 weeks after onset of therapy.
  • Isolation of a Greatwall cDNA and knowledge of its involvement in mitotic processes also facilitates the identification of molecules which decrease Greatwall expression or activity (i.e., Greatwall antagonists).
  • Greatwall expression is measured following the addition of antagonist molecules to the culture medium of Greatwall-expressing cells.
  • the candidate antagonists may be directly administered to animals (for example, zebrafish or mice or rats) and used to screen for antagonists.
  • Greatwall expression is then measured, for example, by standard Northern blot analysis (Ausubel et al., supra) using a Greatwall nucleic acid (or fragment) as a hybridization probe.
  • the level of Greatwall expression in the presence of the candidate molecule is compared to the level measured for the same cells in the same culture medium or test animal, but in the absence of the candidate molecule.
  • Preferred modulators for treating a neoplasia are those which cause a decrease in Greatwall expression.
  • candidate modulators on expression may be measured at the level of Greatwall protein production using the same general approach in combination with standard immunological detection techniques, such as Western blotting or immunoprecipitation with a Greatwall-specific antibody (for example, the Greatwall antibody described herein).
  • useful anti-Greatwall modulators are identified as those which produce a decrease in Greatwall polypeptide production.
  • Candidate modulators may be purified (or substantially purified) molecules or may be one component of a mixture of compounds (e.g., an extract or supernatant obtained from cells).
  • candidate compounds may be screened for those which antagonize native or recombinant Greatwall activity.
  • kinase activity for example, any method described herein
  • a candidate compound is compared to activity in its absence under equivalent conditions.
  • a screen may begin with a pool of candidate compounds, from which one or more useful modulator compounds are isolated in a step-wise fashion.
  • Candidate modulators further may be provided as members of a combinatorial library, which preferably includes synthetic agents prepared according to a plurality of predetermined chemical reactions performed in a plurality of reaction vessels.
  • various starting compounds may be prepared employing one or more of solid-phase synthesis, recorded random mix methodologies and recorded reaction split techniques that permit a given constituent to traceably undergo a plurality of permutations and/or combinations of reaction conditions.
  • the resulting products comprise a library that can be screened followed by iterative selection and synthesis procedures, such as a synthetic combinatorial library of peptides or other compositions that may include small molecules as provided herein.
  • a diverse assortment of such libraries may be prepared according to established procedures, and tested using a Greatwall polypeptide according to the present disclosure.
  • the present invention further provides methods for identifying a molecule that interacts with, or binds to, a Greatwall polypeptide.
  • a molecule generally associates with Greatwall with an affinity constant (Ka) of at least 10 4 , preferably at least 1 10 and most preferably at least 10 .
  • Ka affinity constant
  • Affinity constants may be determined using well known techniques. Methods for identifying interacting molecules may be used, for example, as initial screens for modulating agents, or to identify factors that are involved in the in vivo Greatwall activity. In addition to standard binding assays, there are many other techniques that are well known for identifying interacting molecules, including yeast two-hybrid screens (as described herein), phage display, and affinity techniques. Such techniques may be performed using routine protocols, which are well known to those having ordinary skill in the art.
  • Kinase activity may be measured by any standard assay, for example, it may be measured by monitoring the ability of the enzyme to transfer ⁇ 32P-ATP to a PIP substrate on a TLC plate. If desired, prior to assaying activity, the enzyme may be isolated from a sample, for example, by immunoprecipitation with a Greatwall- specific antibody. The Greatwall mutants described herein have reduced activity in these in vitro assays and may be used as control samples.
  • Candidate Greatwall antagonists include peptide as well as non-peptide molecules (e.g., peptide or non-peptide molecules found, e.g., in a cell extract, mammalian serum, or growth medium on which mammalian cells have been cultured).
  • non-peptide molecules e.g., peptide or non-peptide molecules found, e.g., in a cell extract, mammalian serum, or growth medium on which mammalian cells have been cultured.
  • Antagonists found to be effective at the level of cellular Greatwall expression or activity may be confirmed as useful in animal models (for example, a mouse model sysytem).
  • a molecule which promotes a decrease in Greatwall expression or Greatwall activity is considered particularly useful in the invention; such a molecule may be used, for example, as a therapeutic to decrease the level or activity of native, cellular Greatwall and thereby increases resistance to a neoplasia of the host animal (for example, human).
  • the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the Greatwall polypeptide, such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the Greatwall polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound that is bound to the Greatwall polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • Determining the ability of a test compound to bind to a Greatwall polypeptide also can be accomplished using a technology such as real-time Bimolecular
  • BIA Interaction Analysis
  • BIAcoreTM Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules. It may be desirable to immobilize either the Greatwall polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the Greatwall polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a Greatwall polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • the Greatwall polypeptide is a fusion protein comprising a domain that allows the Greatwall polypeptide to be bound to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed Greatwall polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • a Greatwall polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated Greatwall polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N- hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies which specifically bind to a Greatwall polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the Greatwall polypeptide can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to the Greatwall polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the Greatwall polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds which bind to a Greatwall polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a Greatwall polypeptide or polynucleotide can be used in a cell-based assay system. A Greatwall polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a Greatwall polypeptide or polynucleotide is determined as described above.
  • test compounds can be screened for the ability to bind to Greatwall kinase polypeptides or polynucleotides or to affect Greatwall kinase activity or Greatwall kinase gene expression using high throughput screening (as described herein).
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format. Identification of Test Compounds that Bind to Greatwall Polypeptides
  • Purified Greatwall polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Human Greatwall polypeptides include the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, or 16.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a Greatwall polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 10% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a Greatwall polypeptide. Identification Test Compounds Which Decrease Greatwall Gene Expression
  • test compound is administered to a culture of human cells transfected with a Greatwall polypeptide expression construct and incubated at 37 0 C for 10 to 45 minutes.
  • a culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • RNA is isolated from the two cultures according to standard methods.
  • Northern blots are prepared using total RNA and hybridized with a 32 P -labeled Greatwall polypeptide-specific probe under standard hybridization temperatures.
  • the probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15.
  • a test compound that decreases the Greatwall polypeptide-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of Greatwall gene expression. Identification Test Compounds Which Decrease Greatwall Polypeptide Activity A test compound is administered to a culture of human cells transfected with a
  • a test compound which decreases the Greatwall polypeptide activity of the Greatwall polypeptide relative to the Greatwall polypeptide activity in the absence of the test compound is identified as an inhibitor of Greatwall polypeptide activity.
  • Compounds are assayed for the inhibition of Greatwall according to a variety of conventional screening techniques.
  • Greatwall autophosphorylation is monitored by measuring the amount of ⁇ "32 P incorporated into Greatwall (i.e., autophosphorylation) or into other polypeptide substrates (such as myelin basic protein, casein, Histone Hl, etc) in the presence of a test compound or a combination of test compounds.
  • ⁇ "32 P incorporated into Greatwall i.e., autophosphorylation
  • other polypeptide substrates such as myelin basic protein, casein, Histone Hl, etc
  • test compound or a combination of test compounds such as myelin basic protein, casein, Histone Hl, etc.
  • kinase reaction Greatwall proteins are suspended in 10 ⁇ l of kinase buffer with 100 ⁇ M ATP and 10 ⁇ Ci [ ⁇ 32 P]ATP, and incubated at 30°C for 15 min.
  • neoplasia such as cancer
  • expression is determined in the following tissues: adrenal gland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, uterus, and peripheral blood lymphocytes.
  • Expression in the standard cancer cell lines also is determined. Matched pairs of malignant and normal tissue from the same patient also are tested.
  • the isolation of Greatwall sequences also facilitates the identification of polypeptides which interact with the Greatwall protein.
  • Such polypeptide-encoding sequences are isolated by any standard two hybrid system (see, for example, Fields et al., Nature 340:245-246 (1989); Yang et al., Science 257:680-682 (1992); Zervos et al., Cell 72:223-232 (1993)).
  • all or a part of the Greatwall sequence may be fused to a DNA binding domain (such as the GAL4 or LexA DNA binding domain).
  • a reporter gene for example, a lacZ or LEU2 reporter gene bearing appropriate DNA binding sites, this fusion protein is used as an interaction target.
  • Candidate interacting proteins fused to an activation domain are then co-expressed with the Greatwall fusion in host cells, and interacting proteins are identified by their ability to contact the Greatwall sequence and stimulate reporter gene expression. False positive interactions are eliminated by carrying out a control experiment with an unrelated tester protein fused to an equivalent activation domain (or, if desired, a large panel of such tester proteins).
  • Test compounds can be tested for the ability to increase or decrease the enzymatic activity of a human Greatwall polypeptide.
  • Kinase activity can be measured, for example, as described herein.
  • Enzyme assays can be carried out after contacting either a purified Greatwall polypeptide, a cell membrane preparation, or an intact cell with a test compound.
  • a test compound that decreases an enzymatic activity of a Greatwall polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing Greatwall protein activity.
  • a test compound which increases an enzymatic activity of a human Greatwall polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human Greatwall protein activity.
  • test compounds that increase or decrease Greatwall gene expression are identified.
  • a Greatwall polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the Greatwall polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • the test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of Greatwall mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of a Greatwall polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a Greatwall polypeptide.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell that expresses a Greatwall polynucleotide can be used in a cell- based assay system.
  • the Greatwall polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above.
  • Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used. Diagnostic Methods
  • Greatwall also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding Greatwall polypeptide in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures according to standard methods. Sequence changes at specific locations can also be revealed by standard nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method.
  • the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
  • Altered levels of Greatwall polypeptide also can be detected in various tissues.
  • Assays e.g., an immunological assay
  • Assays used to detect levels of the Greatwall polypeptides in a body sample, or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays. Any of the antibodies disclosed herein are useful for this purpose. Therapeutics
  • a Greatwall antagonist for therapeutic use may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer Greatwall to patients.
  • intravenous administration is preferred, any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracistemal, intraperitoneal, intranasal, aerosol, or oral administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for Greatwall antagonists include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • treatment with a Greatwall antagonist may be combined with any other anti-cancer therapies.
  • Greatwall antagonists may also be used as novel pesticides, for example, to control insects. Because Greatwall controls larval development, compounds which antagonize its action may be used to trigger inappropriately larval development, with a concomitant suspension of or activation cell division. Such pesticides, which target invertebrate Greatwall-mediated events, are useful for enhancing agricultural production.
  • the invention includes any protein which is substantially identical to the Greatwall polypeptide of SEQ E) NO: 2, 4, 6, 8, 10, 12, 14, or 16 such homologs or orthologs include other substantially pure naturally- occurring mammalian Greatwall polypeptides (for example, the human Greatwall polypeptide) as well as allelic variants; natural mutants; induced mutants; proteins encoded by DNA that hybridizes to the Greatwall DNA sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, or 15 under high stringency conditions or, less preferably, under low stringency conditions (e.g., washing at 2X SSC at 4O 0 C with a probe length of at least 40 nucleotides); and proteins specifically bound by antisera directed to a Greatwall polypeptide.
  • such homologs or orthologs include other substantially pure naturally- occurring mammalian Greatwall polypeptides (for example, the human Greatwall polypeptide) as well as allelic variants; natural mutants; induced mutants; proteins encoded by DNA that hybridizes to the Greatwall
  • the invention further includes analogs of any naturally-occurring Greatwall polypeptide.
  • Analogs can differ from the naturally-occurring Greatwall protein by amino acid sequence differences, by post-translational modifications, or by both.
  • Analogs of the invention will generally exhibit at least 50%, more preferably 60%, and most preferably 85% or even 95% identity with a naturally-occurring Greatwall amino acid sequence.
  • Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes.
  • Analogs can also differ from the naturally-occurring Greatwall polypeptide by alterations in primary sequence.
  • the invention also includes Greatwall polypeptide fragments.
  • fragment means at least 20 contiguous amino acids, preferably at least 30 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 or more contiguous amino acids. Fragments of Greatwall polypeptides can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events). Preferable fragments according to the invention include, without limitation, those described herein.
  • all or a portion of the Greatwall polypeptide sequence may be fused to another protein (for example, by recombinant means).
  • Greatwall may be fused to the green fluorescent protein, GFP (Chalfie et al, Science 263:802-805, 1994).
  • GFP green fluorescent protein
  • Such a fusion protein is useful, for example, for monitoring the expression level of Greatwall in vivo (for example, by fluorescence microscopy) following treatment with candidate or known Greatwall antagonists.
  • the methods of the invention may be used to screen for Greatwall modulatory compounds in any mammal, for example, humans, domestic pets, or livestock. Where a non-human mammal is treated or diagnosed, the Greatwall polypeptide, nucleic acid, or antibody employed is preferably specific for that species.

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Abstract

L'invention porte sur des réactifs qui régulent Greatwall, un polypeptide qui fonctionne dans le circuit autorégulateur nécessaire pour générer et maintenir des niveaux élevés du facteur favorisant la maturation, et sur des réactifs qui se lient à Greatwall et qui sont utiles dans la prévention, l'amélioration ou la correction de dysfonctionnements ou maladies tels que, mais pas exclusivement, le cancer.
PCT/US2005/033717 2004-09-17 2005-09-19 Kinases greatwall et leurs utilisations Ceased WO2006034300A2 (fr)

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US61081804P 2004-09-17 2004-09-17
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US60/522,351 2004-09-17
US60/610,818 2004-09-17

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WO2006034300A3 WO2006034300A3 (fr) 2009-04-02

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