Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
MCHKS AS MODIFIERS OF THE CHK1 PATHWAY AND METHODS OF USE
Document Type and Number:
WIPO Patent Application WO/2004/004785
Kind Code:
A1
Abstract:
Human MCHK genes are identified as modulators of the Chk1 pathway, and thus are therapeutic targets for disorders associated with defective Chk1 function. Methods for identifying modulators of Chk1, comprising screening for agents that modulate the activity of MCHK are provided.

Inventors:
FRANCIS-LANG HELEN (US)
ROCHE SIOBHAN (IE)
JOO DANIEL M (US)
NICOLL MONIQUE (US)
HAI BING (US)
ZHANG HAIGUANG (US)
LICKTEIG KIM (US)
AMUNDSEN CRAIG D (US)
JIN YISHENG (US)
ADAMKEWICZ JOANNE I (US)
PLATT DARREN MARK (US)
HAMMONDS R GLENN JR (US)
Application Number:
PCT/US2003/021379
Publication Date:
January 15, 2004
Filing Date:
July 09, 2003
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
EXELIXIS INC (US)
FRANCIS-LANG HELEN (US)
ROCHE SIOBHAN (IE)
JOO DANIEL M (US)
NICOLL MONIQUE (US)
HAI BING (US)
ZHANG HAIGUANG (US)
LICKTEIG KIM (US)
AMUNDSEN CRAIG D (US)
JIN YISHENG (US)
ADAMKEWICZ JOANNE I (US)
PLATT DARREN MARK (US)
HAMMONDS R GLENN JR (US)
International Classes:
A01K67/033; A61K49/00; C12N9/12; C12Q1/68; (IPC1-7): A61K49/00; A01K67/00; A01K67/033; C12Q1/68; G01N33/00
Domestic Patent References:
WO1999011795A11999-03-11
Other References:
LIU Q.: "Chk1 is an essential kinase that is regulated by Atr and required for the g2/m DNA damage checkpoint", GENES AND DEVELOPMENT, vol. 14, 2000, pages 1448 - 1459, XP002972629
SENDEROWICZ A.M.: "Small molecule modulators for cyclin-dependent kinases for cancer therapy", ONCOGENE, vol. 19, 2000, pages 6600 - 6606, XP002972630
TAKAI H.: "Aberrant cell cycle checkpoint function and early embryonic death in Chk1-/- mice", GENES AND DEVELOPMENT, vol. 14, 2000, pages 1439 - 1447, XP002907710
Attorney, Agent or Firm:
Shayesteh, Laleh (Inc. P. O. Box 511, 170 Harbor Wa, South San Francisco CA, US)
Download PDF:
Claims:
WHAT IS CLAIMED IS:
1. A method of identifying a candidate Chkl pathway modulating agent, said method comprising the steps of: (a) providing an assay system comprising a MCHK polypeptide or nucleic acid; (b) contacting the assay system with a test agent under conditions whereby, but for the presence of the test agent, the system provides a reference activity; and (c) detecting a test agentbiased activity of the assay system, wherein a difference between the test agentbiased activity and the reference activity identifies the test agent as a candidate Chkl pathway modulating agent.
2. The method of Claim 1 wherein the assay system comprises cultured cells that express the MCHK polypeptide.
3. The method of Claim 2 wherein the cultured cells additionally have defective Chkl function.
4. The method of Claim 1 wherein the assay system includes a screening assay comprising a MCHK polypeptide, and the candidate test agent is a small molecule modulator.
5. The method of Claim 4 wherein the assay is a binding assay.
6. The method of Claim 1 wherein the assay system is selected from the group consisting of an apoptosis assay system, a cell proliferation assay system, an angiogenesis assay system, and a hypoxic induction assay system.
7. The method of Claim 1 wherein the assay system includes a binding assay comprising a MCHK polypeptide and the candidate test agent is an antibody.
8. The method of Claim 1 wherein the assay system includes an expression assay comprising a MCHK nucleic acid and the candidate test agent is a nucleic acid modulator.
9. The method of Claim 8 wherein the nucleic acid modulator is an antisense oligomer.
10. The method of Claim 8 wherein the nucleic acid modulator is a PMO.
11. The method of Claim 1 additionally comprising: (d) administering the candidate Chkl pathway modulating agent identified in (c) to a model system comprising cells defective in Chkl function and, detecting a phenotypic change in the model system that indicates that the Chkl function is restored.
12. The method of Claim 11 wherein the model system is a mouse model with defective Chkl function.
13. A method for modulating a Chkl pathway of a cell comprising contacting a cell defective in Chkl function with a candidate modulator that specifically binds to a MCHK polypeptide, whereby Chkl function is restored.
14. The method of Claim 13 wherein the candidate modulator is administered to a vertebrate animal predetermined to have a disease or disorder resulting from a defect in Chkl function.
15. The method of Claim 13 wherein the candidate modulator is selected from the group consisting of an antibody and a small molecule.
16. The method of Claim 1, comprising the additional steps of: (d) providing a secondary assay system comprising cultured cells or a nonhuman animal expressing MCHK, (e) contacting the secondary assay system with the test agent of (b) or an agent derived therefrom under conditions whereby, but for the presence of the test agent or agent derived therefrom, the system provides a reference activity; and (f) detecting an agentbiased activity of the second assay system, wherein a difference between the agentbiased activity and the reference activity of the second assay system confirms the test agent or agent derived therefrom as a candidate Chkl pathway modulating agent, and wherein the second assay detects an agentbiased change in the Chkl pathway.
17. The method of Claim 16 wherein the secondary assay system comprises cultured cells.
18. The method of Claim 16 wherein the secondary assay system comprises a nonhuman animal.
19. The method of Claim 18 wherein the nonhuman animal misexpresses a Chkl pathway gene.
20. A method of modulating Chkl pathway in a mammalian cell comprising contacting the cell with an agent that specifically binds a MCHK polypeptide or nucleic acid.
21. The method of Claim 20 wherein the agent is administered to a mammalian animal predetermined to have a pathology associated with the Chkl pathway.
22. The method of Claim 20 wherein the agent is a small molecule modulator, a nucleic acid modulator, or an antibody.
23. A method for diagnosing a disease in a patient comprising: (a) obtaining a biological sample from the patient; (b) contacting the sample with a probe for MCHK expression; (c) comparing results from step (b) with a control; (d) determining whether step (c) indicates a likelihood of disease.
24. The method of claim 23 wherein said disease is cancer.
Description:
MCHKS AS MODIFIERS OF THE CHK1 PATHWAY AND METHODS OF USE REFERENCE TO RELATED APPLICATIONS This application claims priority to U. S. provisional patent applications 60/394,845 filed 7/10/2002 and 60/410,986 filed 9/16/2002. The contents of the prior applications are hereby incorporated in their entirety.

BACKGROUND OF THE INVENTION The integrity of the genome is monitored by cell cycle checkpoints that, in response to DNA damage, delay progression through the cell cycle until the damage has been repaired. Chkl kinase is an essential component of the G2 DNA damage checkpoint (Liu et. al. Genes Dev (2000) 14: 1448-1459, Takai et. al. Genes Dev (2000) 14: 1439- 1447). Specifically, Chkl is activated by the DNA damage sensor, ATR, and the checkpoint Rad proteins in response to genotoxic stress. The direct downstream target of the Chkl kinase is the Cdc25C phosphatase (Sanchez et. al. Science (1997) 277: 1497- 1501). Cdc25C promotes progression through the G2/M phase of the cell cycle by removing the inhibitory phosphate groups (Thrl4 and Tyrl5) from Cdc2, the cyclin- dependent kinase that promotes mitosis when bound to cycB. Phosphorylation of Cdc25C by Chkl directly inihibits its phosphatase activity and creates a binding site for 14-3-3 proteins resulting in its export from the nucleus (Peng et. al. Science (1997) 277: 1501- 1505). The result of the inhibitory phosphorylation of Cdc25C is that Cdc2/cycB remains in the inactive phosphorylated state and a G2 cell cycle arrest occurs.

Chkl can also cause a G1 cell cycle arrest or apoptosis by phosphorylating and stabilizing p53 (Shieh et. al. Genes Dev. (2000) 14: 289-300, Chehab et. al. Genes Dev.

(200) 14,278-288). The p53 gene is one of the most commonly found mutations in cancer cells and is an essential component of the G1 cell cycle checkpoint (Levine Cell (1997) 88: 323-331; Hollstein et. al. Nucleic Acids Res. (1994) 22: 3551-3555). Indeed, more than 90% of solid tumors contain a defective Gl DNA damage checkpoint. Studies have shown that p53-deficient tumor cells are more susceptible to the cytotoxic effects of DNA damaging agents if the G2 checkpoint is also disrupted by inhibiting either ATR or Chkl (Nghiem et. al. PNAS (2001) 98: 9092-9097, Suganuma et. al. Cancer Res (1999) 59: 5887- 5891). The Chkl kinase inhibitor, UCN-01 is currently undergoing clinical trials as a modulator of anti-cancer drug sensitivity (Busby et. al. Cancer Res (2000) 60: 2108-2102).

Therefore, other essential components of the G2 DNA damage checkpoint may also be

effective drug targets for selectively killing Gl checkpoint defective cancer cells is response to chemotherapeutic DNA damaging agents. Chkl sequences are highly conserved in evolution, and have been identified in a number of organisms including yeast (Walworth, N. , et al (1993) Nature 363: 368-371), Drosophila (Fogarty, P. , et al. (1997) Curr. Biol. 7: 418-426), mouse (Sanchez, Y, et al (1997) Science 277: 1497-1501), and human (Sanchez, Y. , et al (1997) Science 277: 1497-1501), among others.

The ability to manipulate the genomes of model organisms such as Drosophila provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms.

Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Mechler BM et al. , 1985 EMBO J 4: 1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33- 74; Watson KL., et al. , 1994 J Cell Sci. 18 : 19-33; Miklos GL, and Rubin GM. 1996 Cell 86 : 521-529; Wassarman DA, et al. , 1995 Curr Opin Gen Dev 5: 44-50; and Booth DR.

1999 Cancer Metastasis Rev. 18: 261-284). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e. g. knockout) or overexpression of a gene (referred to as genetic entry point") that yields a visible phenotype. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a"modifier"involved in the same or overlapping pathway as the genetic entry point. When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as Chkl, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.

All references cited herein, including patents, patent applications, publications, and sequence information in referenced Genbank identifier numbers, are incorporated herein in their entireties.

SUMMARY OF THE INVENTION We have discovered genes that modify the Chkl pathway in Drosophila, and identified their human orthologs, hereinafter referred to as modifier of Chkl (MCHK).

The invention provides methods for utilizing these Chkl modifier genes and polypeptides

to identify MCHK-modulating agents that are candidate therapeutic agents that can be used in the treatment of disorders associated with defective or impaired Chkl function and/or MCHK function. Preferred MCHK-modulating agents specifically bind to MCHK polypeptides and restore Chkl function. Other preferred MCHK-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress MCHK gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i. e. DNA or mRNA).

MCHK modulating agents may be evaluated by any convenient ill vitro or in vivo assay for molecular interaction with an MCHK polypeptide or nucleic acid. In one embodiment, candidate MCHK modulating agents are tested with an assay system comprising a MCHK polypeptide or nucleic acid. Agents that produce a change in the activity of the assay system relative to controls are identified as candidate Chkl modulating agents. The assay system may be cell-based or cell-free. MCHK-modulating agents include MCHK related proteins (e. g. dominant negative mutants, and biotherapeutics); MCHK-specific antibodies ; MCHK-specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind to or interact with MCHK or compete with MCHK binding partner (e. g. by binding to an MCHK binding partner). In one specific embodiment, a small molecule modulator is identified using a binding assay. In specific embodiments, the screening assay system is selected from an apoptosis assay, a cell proliferation assay, an angiogenesis assay, and a hypoxic induction assay.

In another embodiment, candidate Chkl pathway modulating agents are further tested using a second assay system that detects changes in the Chkl pathway, such as angiogenic, apoptotic, or cell proliferation changes produced by the originally identified candidate agent or an agent derived from the original agent. The second assay system may use cultured cells or non-human animals. In specific embodiments, the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the Chkl pathway, such as an angiogenic, apoptotic, or cell proliferation disorder (e. g. cancer).

The invention further provides methods for modulating the MCHK function and/or the Chkl pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a MCHK polypeptide or nucleic acid. The agent may be a small molecule modulator, a nucleic acid modulator, or an antibody and may be administered to a mammalian animal predetermined to have a pathology associated the Chkl pathway.

DETAILED DESCRIPTION OF THE INVENTION Genetic screens were designed to identify modifiers of the Chkl pathway in Drosophila, where the Chkl gene was overexpressed specifically in the eye, resulting in a G2 cell cycle arrest and a deterioration of general eye morphology. The screen was designed to identify suppressors and enhancers of Drosophila Chkl. Modifiers of the Chkl pathway were identified, followed by identification of their orthologs. Accordingly, vertebrate orthologs of these modifiers, and preferably the human orthologs, MCHK genes (i. e. , nucleic acids and polypeptides) are attractive drug targets for the treatment of pathologies associated with a defective Chkl signaling pathway, such as cancer. Table 1 (Example II) lists the modifiers and their orthologs.

In vitro and in vivo methods of assessing MCHK function are provided herein.

Modulation of the MCHK or their respective binding partners is useful for understanding the association of the Chkl pathway and its members in normal and disease conditions and for developing diagnostics and therapeutic modalities for Chkl related pathologies.

MCHK-modulating agents that act by inhibiting or enhancing MCHK expression, directly or indirectly, for example, by affecting an MCHK function such as enzymatic (e. g., catalytic) or binding activity, can be identified using methods provided herein. MCHK modulating agents are useful in diagnosis, therapy and pharmaceutical development.

Nucleic acids and polypeptides of the invention Sequences related to MCHK nucleic acids and polypeptides that can be used in the invention are disclosed in Genbank (referenced by Genbank identifier (GI) or RefSeq number), shown in Table 1 and in the appended sequence listing.

The term"MCHK polypeptide"refers to a full-length MCHK protein or a functionally active fragment or derivative thereof. A"functionally active"MCHK fragment or derivative exhibits one or more functional activities associated with a full- length, wild-type MCHK protein, such as antigenic or immunogenic activity, enzymatic activity, ability to bind natural cellular substrates, etc. The functional activity of MCHK proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al., eds. , John Wiley & Sons, Inc. , Somerset, New Jersey) and as further discussed below. In one embodiment, a functionally active MCHK polypeptide is a MCHK derivative capable of rescuing defective endogenous MCHK activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species. For purposes herein,

functionally active fragments also include those fragments that comprise one or more structural domains of an MCHK, such as a binding domain. Protein domains can be identified using the PFAM program (Bateman A. , et al. , Nucleic Acids Res, 1999,27 : 260- 2). Methods for obtaining MCHK polypeptides are also further described below. In some embodiments, preferred fragments are functionally active, domain-containing fragments comprising at least 25 contiguous amino acids, preferably at least 50, more preferably 75, and most preferably at least 100 contiguous amino acids of an MCHK. In further preferred embodiments, the fragment comprises the entire functionally active domain.

The term"MCHK nucleic acid"refers to a DNA or RNA molecule that encodes a MCHK polypeptide. Preferably, the MCHK polypeptide or nucleic acid or fragment thereof is from a human, but can also be an ortholog, or derivative thereof with at least 70% sequence identity, preferably at least 80%, more preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with human MCHK.

Methods of identifying orthlogs are known in the art. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3- dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences. Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95: 5849-5856 ; Huynen MA et al., Genome Research (2000) 10: 1204-1210). Programs for multiple sequence alignment, such as CLUSTAL (Thompson JD et al, 1994, Nucleic Acids Res 22: 4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees. In a phylogenetic tree representing multiple homologous sequences from diverse species (e. g. , retrieved through BLAST analysis), orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species. Structural threading or other analysis of protein folding (e. g. , using software by ProCeryon, Biosciences, Salzburg, Austria) may also identify potential orthologs. In evolution, when a gene duplication event follows speciation, a single gene in one species, such as Drosophila, may correspond to multiple genes (paralogs) in another, such as human. As used herein, the term"orthologs"encompasses paralogs. As used herein, "percent (%) sequence identity"with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject

sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2. 0al9 (Altschul et al., J. Mol. Biol. (1997) 215: 403-410) with all the search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A % identity value is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. "Percent (%) amino acid sequence similarity"is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.

A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.

Alternatively, an alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances in Applied Mathematics 2: 482-489; database: European Bioinformatics Institute; Smith and Waterman, 1981, J. of Molec. Biol. , 147: 195-197; Nicholas et al., 1998,"A Tutorial on Searching Sequence Databases and Sequence Scoring Methods" (www. psc. edu) and references cited therein.; W. R. Pearson, 1991, Genomics 11: 635-650). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3: 353- 358, National Biomedical Research Foundation, Washington, D. C. , USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res. 14 (6): 6745-6763). The Smith-Waterman algorithm may be employed where default parameters are used for scoring (for example, gap open penalty of 12, gap extension penalty of two). From the data generated, the "Match"value reflects"sequence identity."

Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of an MCHK. The stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are set out in readily available procedure texts (e. g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989) ). In some embodiments, a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of an MCHK under high stringency hybridization conditions that are: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C in a solution comprising 6X single strength citrate (SSC) (1X SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0. 05% sodium pyrophosphate and 100 yg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, 1X Denhardt's solution, 100 yg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C for 1h in a solution containing 0. 1X SSC and 0.1% SDS (sodium dodecyl sulfate).

In other embodiments, moderately stringent hybridization conditions are used that are: pretreatment of filters containing nucleic acid for 6 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500, ug/ml denatured salmon sperm DNA; hybridization for 18-20h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCI (pH7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0. 2% BSA, 100 All salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C in a solution containing 2X SSC and 0.1% SDS.

Alternatively, low stringency conditions can be used that are: incubation for 8 hours to overnight at 37° C in a solution comprising 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7. 6), 5X Denhardt's solution, 10% dextran sulfate, and 20 yg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 x SSC at about 37° C for 1 hour.

Isolation, Production, Expression, and Mis-expression of MCHK Nucleic Acids and Polypeptides MCHK nucleic acids and polypeptides, are useful for identifying and testing agents that modulate MCHK function and for other applications related to the involvement of

MCHK in the Chkl pathway. MCHK nucleic acids and derivatives and orthologs thereof may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR) are well known in the art. In general, the particular use for the protein will dictate the particulars of expression, production, and purification methods.

For instance, production of proteins for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of proteins for antibody generation may require structural integrity of particular epitopes. Expression of proteins to be purified for screening or antibody production may require the addition of specific tags (e. g. , generation of fusion proteins). Overexpression of an MCHK protein for assays used to assess MCHK function, such as involvement in cell cycle regulation or hypoxic response, may require expression in eukaryotic cell lines capable of these cellular activities. Techniques for the expression, production, and purification of proteins are well known in the art; any suitable means therefore may be used (e. g. , Higgins SJ and Hames BD (eds. ) Protein Expression: A Practical Approach, Oxford University Press Inc. , New York 1999 ; Stanbury PF et al. , Principles of Fermentation Technology, 2 edition, Elsevier Science, New York, 1995; Doonan S (ed.) Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan JE et al, Current Protocols in Protein Science (eds. ), 1999, John Wiley & Sons, New York). In particular embodiments, recombinant MCHK is expressed in a cell line known to have defective Chkl function. The recombinant cells are used in cell-based screening assay systems of the invention, as described further below.

The nucleotide sequence encoding an MCHK polypeptide can be inserted into any appropriate expression vector. The necessary transcriptional and translational signals, including promoter/enhancer element, can derive from the native MCHK gene and/or its flanking regions or can be heterologous. A variety of host-vector expression systems may be utilized, such as mammalian cell systems infected with virus (e. g. vaccinia virus, adenovirus, etc.) ; insect cell systems infected with virus (e. g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, plasmid, or cosmid DNA. An isolated host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.

To detect expression of the MCHK gene product, the expression vector can comprise a promoter operably linked to an MCHK gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e. g. thymidine kinase activity,

resistance to antibiotics, etc.). Alternatively, recombinant expression vectors can be identified by assaying for the expression of the MCHK gene product based on the physical or functional properties of the MCHK protein in in vitro assay systems (e. g. immunoassays).

The MCHK protein, fragment, or derivative may be optionally expressed as a fusion, or chimeric protein product (i. e. it is joined via a peptide bond to a heterologous protein sequence of a different protein), for example to facilitate purification or detection.

A chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other using standard methods and expressing the chimeric product. A chimeric product may also be made by protein synthetic techniques, e. g. by use of a peptide synthesizer (Hunkapiller et al., Nature (1984) 310: 105-111).

Once a recombinant cell that expresses the MCHK gene sequence is identified, the gene product can be isolated and purified using standard methods (e. g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis). Alternatively, native MCHK proteins can be purified from natural sources, by standard methods (e. g. immunoaffinity purification). Once a protein is obtained, it may be quantified and its activity measured by appropriate methods, such as immunoassay, bioassay, or other measurements of physical properties, such as crystallography.

The methods of this invention may also use cells that have been engineered for altered expression (mis-expression) of MCHK or other genes associated with the Chkl pathway. As used herein, mis-expression encompasses ectopic expression, over- expression, under-expression, and non-expression (e. g. by gene knock-out or blocking expression that would otherwise normally occur).

Genetically modified animals Animal models that have been genetically modified to alter MCHK expression may be used in in vivo assays to test for activity of a candidate Chkl modulating agent, or to further assess the role of MCHK in a Chkl pathway process such as apoptosis or cell proliferation. Preferably, the altered MCHK expression results in a detectable phenotype, such as decreased or increased levels of cell proliferation, angiogenesis, or apoptosis compared to control animals having normal MCHK expression. The genetically modified animal may additionally have altered Chkl expression (e. g. Chkl knockout). Preferred

genetically modified animals are mammals such as primates, rodents (preferably mice or rats), among others. Preferred non-mammalian species include zebrafish, C. elegans, and Drosophila. Preferred genetically modified animals are transgenic animals having a heterologous nucleic acid sequence present as an extrachromosomal element in a portion of its cells, i. e. mosaic animals (see, for example, techniques described by Jakobovits, 1994, Curr. Biol. 4: 761-763. ) or stably integrated into its germ line DNA (i. e. , in the genomic sequence of most or all of its cells). Heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal.

Methods of making transgenic animals are well-known in the art (for transgenic mice see Brinster et al. , Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985), U. S. Pat. Nos.

4,736, 866 and 4,870, 009, both by Leder et al. , U. S. Pat. No. 4, 873, 191 by Wagner et al., and Hogan, B. , Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., (1986) ; for particle bombardment see U. S. Pat. No. , 4,945, 050, by Sandford et al. ; for transgenic Drosophila see Rubin and Spradling, Science (1982) 218: 348-53 and U. S. Pat. No. 4,670, 388; for transgenic insects see Berghammer A. J. et al., A Universal Marker for Transgenic Insects (1999) Nature 402: 370-371 ; for transgenic Zebrafish see Lin S. , Transgenic Zebrafish, Methods Mol Biol. (2000); 136: 375-3830); for microinjection procedures for fish, amphibian eggs and birds see Houdebine and Chourrout, Experientia (1991) 47: 897-905; for transgenic rats see Hammer et al., Cell (1990) 63: 1099-1112; and for culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct injection see, e. g. , Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed. , IRL Press (1987) ). Clones of the nonhuman transgenic animals can be produced according to available methods (see Wilmut, 1. et al. (1997) Nature 385: 810-813 ; and PCT International Publication Nos. WO 97/07668 and WO 97/07669).

In one embodiment, the transgenic animal is a"knock-out"animal having a heterozygous or homozygous alteration in the sequence of an endogenous MCHK gene that results in a decrease of MCHK function, preferably such that MCHK expression is undetectable or insignificant. Knock-out animals are typically generated by homologous recombination with a vector comprising a transgene having at least a portion of the gene to be knocked out. Typically a deletion, addition or substitution has been introduced into the transgene to functionally disrupt it. The transgene can be a human gene (e. g. , from a

human genomic clone) but more preferably is an ortholog of the human gene derived from the transgenic host species. For example, a mouse MCHK gene is used to construct a homologous recombination vector suitable for altering an endogenous MCHK gene in the mouse genome. Detailed methodologies for homologous recombination in mice are available (see Capecchi, Science (1989) 244: 1288-1292 ; Joyner et al., Nature (1989) 338 : 153-156). Procedures for the production of non-rodent transgenic mammals and other animals are also available (Houdebine and Chourrout, supra ; Pursel et al., Science (1989) 244: 1281-1288 ; Simms et al., Bio/Technology (1988) 6: 179-183). In a preferred embodiment, knock-out animals, such as mice harboring a knockout of a specific gene, may be used to produce antibodies against the human counterpart of the gene that has been knocked out (Claesson MH et al., (1994) Scan J Immunol 40: 257-264; Declerck PJ et al. , (1995) J Biol Chem. 270: 8397-400).

In another embodiment, the transgenic animal is a"knock-in"animal having an alteration in its genome that results in altered expression (e. g. , increased (including ectopic) or decreased expression) of the MCHK gene, e. g. , by introduction of additional copies of MCHK, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the MCHK gene. Such regulatory sequences include inducible, tissue-specific, and constitutive promoters and enhancer elements. The knock-in can be homozygous or heterozygous.

Transgenic nonhuman animals can also be produced that contain selected systems allowing for regulated expression of the transgene. One example of such a system that may be produced is the cre/loxP recombinase system of bacteriophage PI (Lakso et al., PNAS (1992) 89 : 6232-6236; U. S. Pat. No. 4,959, 317). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of"double"transgenic animals, e. g. , by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.

(1991) Science 251: 1351-1355; U. S. Pat. No. 5,654, 182). In a preferred embodiment, both Cre-LoxP and Flp-Frt are used in the same system to regulate expression of the transgene, and for sequential deletion of vector sequences in the same cell (Sun X et al (2000) Nat Genet 25: 83-6).

The genetically modified animals can be used in genetic studies to further elucidate the Chkl pathway, as animal models of disease and disorders implicating defective Chkl function, and for in vivo testing of candidate therapeutic agents, such as those identified in screens described below. The candidate therapeutic agents are administered to a genetically modified animal having altered MCHK function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered MCHK expression that receive candidate therapeutic agent.

In addition to the above-described genetically modified animals having altered MCHK function, animal models having defective Chkl function (and otherwise normal MCHK function), can be used in the methods of the present invention. For example, a Chkl knockout mouse can be used to assess, in vivo, the activity of a candidate Chkl modulating agent identified in one of the in vitro assays described below. Preferably, the candidate Chkl modulating agent when administered to a model system with cells defective in Chkl function, produces a detectable phenotypic change in the model system indicating that the Chkl function is restored, i. e. , the cells exhibit normal cell cycle progression.

Modulating Agents The invention provides methods to identify agents that interact with and/or modulate the function of MCHK and/or the Chkl pathway. Modulating agents identified by the methods are also part of the invention. Such agents are useful in a variety of diagnostic and therapeutic applications associated with the Chkl pathway, as well as in further analysis of the MCHK protein and its contribution to the Chkl pathway.

Accordingly, the invention also provides methods for modulating the Chkl pathway comprising the step of specifically modulating MCHK activity by administering a MCHK- interacting or-modulating agent.

As used herein, an"MCHK-modulating agent"is any agent that modulates MCHK function, for example, an agent that interacts with MCHK to inhibit or enhance MCHK activity or otherwise affect normal MCHK function. MCHK function can be affected at any level, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In a preferred embodiment, the MCHK-modulating agent specifically modulates the function of the MCHK. The phrases"specific modulating agent", "specifically modulates", etc. , are used herein to refer to modulating agents that

directly bind to the MCHK polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the MCHK. These phrases also encompass modulating agents that alter the interaction of the MCHK with a binding partner, substrate, or cofactor (e. g. by binding to a binding partner of an MCHK, or to a protein/binding partner complex, and altering MCHK function). In a further preferred embodiment, the MCHK- modulating agent is a modulator of the Chkl pathway (e. g. it restores and/or upregulates Chkl function) and thus is also a Chkl-modulating agent.

Preferred MCHK-modulating agents include small molecule compounds; MCHK- interacting proteins, including antibodies and other biotherapeutics; and nucleic acid modulators such as antisense and RNA inhibitors. The modulating agents may be formulated in pharmaceutical compositions, for example, as compositions that may comprise other active ingredients, as in combination therapy, and/or suitable carriers or excipients. Techniques for formulation and administration of the compounds may be found in"Remington's Pharmaceutical Sciences"Mack Publishing Co. , Easton, PA, 19th edition.

Small molecule modulators Small molecules are often preferred to modulate function of proteins with enzymatic function, and/or containing protein interaction domains. Chemical agents, referred to in the art as"small molecule"compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably less than 500 daltons. This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based on known or inferred properties of the MCHK protein or may be identified by screening compound libraries. Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for MCHK- modulating activity. Methods for generating and obtaining compounds are well known in the art (Schreiber SL, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000) 151: 1947-1948).

Small molecule modulators identified from screening assays, as described below, can be used as lead compounds from which candidate clinical compounds may be designed, optimized, and synthesized. Such clinical compounds may have utility in

treating pathologies associated with the Chkl pathway. The activity of candidate small molecule modulating agents may be improved several-fold through iterative secondary functional validation, as further described below, structure determination, and candidate modulator modification and testing. Additionally, candidate clinical compounds are generated with specific regard to clinical and pharmacological properties. For example, the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.

Protein Modulators Specific MCHK-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the Chkl pathway and related disorders, as well as in validation assays for other MCHK-modulating agents. In a preferred embodiment, MCHK-interacting proteins affect normal MCHK function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In another embodiment, MCHK-interacting proteins are useful in detecting and providing information about the function of MCHK proteins, as is relevant to Chkl related disorders, such as cancer (e. g. , for diagnostic means).

An MCHK-interacting protein may be endogenous, i. e. one that naturally interacts genetically or biochemically with an MCHK, such as a member of the MCHK pathway that modulates MCHK expression, localization, and/or activity. MCHK-modulators include dominant negative forms of MCHK-interacting proteins and of MCHK proteins themselves. Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous MCHK-interacting proteins (Finley, R. L. et al. (1996) in DNA Cloning- Expression Systems: A Practical Approach, eds. Glover D. & Hames B. D (Oxford University Press, Oxford, England), pp. 169-203; Fashema SF et al. , Gene (2000) 250: 1- 14; Drees BL Curr Opin Chem Biol (1999) 3: 64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27: 919-29; and U. S. Pat. No. 5,928, 868). Mass spectrometry is an alternative preferred method for the elucidation of protein complexes (reviewed in, e. g. , Pandley A and Mann M, Nature (2000) 405: 837-846; Yates JR 3rd, Trends Genet (2000) 16: 5-8).

An MCHK-interacting protein may be an exogenous protein, such as an MCHK- specific antibody or a T-cell antigen receptor (see, e. g. , Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using antibodies: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). MCHK antibodies are further discussed below.

In preferred embodiments, an MCHK-interacting protein specifically binds an MCHK protein. In alternative preferred embodiments, an MCHK-modulating agent binds an MCHK substrate, binding partner, or cofactor.

Antibodies In another embodiment, the protein modulator is an MCHK specific antibody agonist or antagonist. The antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify MCHK modulators. The antibodies can also be used in dissecting the portions of the MCHK pathway responsible for various cellular responses and in the general processing and maturation of the MCHK.

Antibodies that specifically bind MCHK polypeptides can be generated using known methods. Preferably the antibody is specific to a mammalian ortholog of MCHK polypeptide, and more preferably, to human MCHK. Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab'). sub. 2 fragments, fragments produced by a FAb expression library, anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

Epitopes of MCHK which are particularly antigenic can be selected, for example, by routine screening of MCHK polypeptides for antigenicity or by applying a theoretical method for selecting antigenic regions of a protein (Hopp and Wood (1981), Proc. Nati.

Acad. Sci. U. S. A. 78: 3824-28; Hopp and Wood, (1983) Mol. Immunol. 20: 483-89 ; Sutcliffe et al., (1983) Science 219: 660-66) to the amino acid sequence of an MCHK.

Monoclonal antibodies with affinities of 108 lvf 1 preferably 109 M-'to to 10l° M-l, or stronger can be made by standard procedures as described (Harlow and Lane, supra ; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U. S. Pat. Nos. 4, 381, 292; 4,451, 570; and 4,618, 577). Antibodies may be generated against crude cell extracts of MCHK or substantially purified fragments thereof.

If MCHK fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an MCHK protein. In a particular embodiment, MCHK-specific antigens and/or immunogens are coupled to carrier proteins that stimulate the immune response. For example, the subject polypeptides are covalently coupled to the keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant, which enhances the immune response. An appropriate immune system such as a laboratory rabbit or mouse is immunized according to conventional protocols.

The presence of MCHK-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding MCHK polypeptides. Other assays, such as radioimmunoassays or fluorescent assays might also be used.

Chimeric antibodies specific to MCHK polypeptides can be made that contain different portions from different animal species. For instance, a human immunoglobulin constant region may be linked to a variable region of a murine mAb, such that the antibody derives its biological activity from the human antibody, and its binding specificity from the murine fragment. Chimeric antibodies are produced by splicing together genes that encode the appropriate regions from each species (Morrison et al., Proc. Natl. Acad. Sci. (1984) 81 : 6851-6855; Neuberger et al. , Nature (1984) 312: 604-608; Takeda et al. , Nature (1985) 31: 452-454). Humanized antibodies, which are a form of chimeric antibodies, can be generated by grafting complementary-determining regions (CDRs) (Carlos, T. M. , J. M. Harlan. 1994. Blood 84 : 2068-2101) of mouse antibodies into a background of human framework regions and constant regions by recombinant DNA technology (Riechmann LM, et al., 1988 Nature 323: 323-327). Humanized antibodies contain-10% murine sequences and-90% human sequences, and thus further reduce or eliminate immunogenicity, while retaining the antibody specificities (Co MS, and Queen C. 1991 Nature 351: 501-501 ; Morrison SL. 1992 Ann. Rev. Immun.

10: 239-265). Humanized antibodies and methods of their production are well-known in the art (U. S. Pat. Nos. 5,530, 101,5, 585, 089, 5,693, 762, and 6,180, 370).

MCHK-specific single chain antibodies which are recombinant, single chain polypeptides formed by linking the heavy and light chain fragments of the Fv regions via an amino acid bridge, can be produced by methods known in the art (U. S. Pat. No.

4,946, 778; Bird, Science (1988) 242: 423-426; Huston et al. , Proc. Natl. Acad. Sci. USA (1988) 85: 5879-5883; and Ward et al. , Nature (1989) 334: 544-546).

Other suitable techniques for antibody production involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors (Huse et al., Science (1989) 246: 1275-1281). As used herein, T-cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).

The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, antibodies will be labeled by joining, either covalently or non-covalently, a substance that provides for a detectable signal, or that is toxic to cells

that express the targeted protein (Menard S, et al. , Int J. Biol Markers (1989) 4: 131-134).

A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorescent emitting lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (U. S. Pat. Nos. 3, 817, 837; 3,850, 752; 3,939, 350; 3,996, 345; 4,277, 437; 4,275, 149; and 4,366, 241). Also, recombinant immunoglobulins may be produced (U. S. Pat. No. 4,816, 567). Antibodies to cytoplasmic polypeptides may be delivered and reach their targets by conjugation with membrane-penetrating toxin proteins (U. S. Pat. No. 6,086, 900).

When used therapeutically in a patient, the antibodies of the subject invention are typically administered parenterally, when possible at the target site, or intravenously. The therapeutically effective dose and dosage regimen is determined by clinical studies.

Typically, the amount of antibody administered is in the range of about 0.1 mg/kg-to about 10 mg/kg of patient weight. For parenteral administration, the antibodies are formulated in a unit dosage injectable form (e. g. , solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle. Such vehicles are inherently nontoxic and non-therapeutic. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used. The vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential. The antibodies'concentrations in such vehicles are typically in the range of about 1 mg/ml to aboutIO mg/ml.

Immunotherapeutic methods are further described in the literature (US Pat. No. 5,859, 206; W00073469).

Nucleic Acid Modulators Other preferred MCHK-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit MCHK activity. Preferred nucleic acid modulators interfere with the function of the MCHK nucleic acid such as DNA replication, transcription, translocation of the MCHK RNA to the site of protein translation, translation of protein from the MCHK RNA, splicing of the MCHK RNA to yield one or more mRNA species, or catalytic activity which may be engaged in or facilitated by the MCHK RNA.

In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to an MCHK mRNA to bind to and prevent translation, preferably by binding to the 5'untranslated region. MCHK-specific antisense oligonucleotides, preferably range from at least 6 to about 200 nucleotides. In some embodiments the oligonucleotide is preferably at least 10,15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably less than 50,40, or 30 nucleotides in length. The oligonucleotide can be DNA or RNA or a chimeric mixture or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, agents that facilitate transport across the cell membrane, hybridization-triggered cleavage agents, and intercalating agents.

In another embodiment, the antisense oligomer is a phosphothioate morpholino oligomer (PMO). PMOs are assembled from four different morpholino subunits, each of which contain one of four genetic bases (A, C, G, or T) linked to a six-membered morpholine ring. Polymers of these subunits are joined by non-ionic phosphodiamidate intersubunit linkages. Details of how to make and use PMOs and other antisense oligomers are well known in the art (e. g. see W099/18193 ; Probst JC, Antisense Oligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22 (3): 271-281 ; Summerton J, and Weller D. 1997 Antisense Nucleic Acid Drug Dev.: 7: 187-95; US Pat. No.

5,235, 033; and US Pat No. 5,378, 841).

Alternative preferred MCHK nucleic acid modulators are double-stranded RNA species mediating RNA interference (RNAi). RNAi is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and humans are known in the art (Fire A, et al. , 1998 Nature 391: 806-811 ; Fire, A. Trends Genet. 15,358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M. , et al., Nature Rev. Genet. 2,110-1119 (2001) ; Tuschl, T. Chem. Biochem. 2, 239- 245 (2001); Hamilton, A. et al. , Science 286,950-952 (1999); Hammond, S. M. , et al., Nature 404,293-296 (2000); Zamore, P. D. , et al. , Cell 101,25-33 (2000); Bernstein, E., et al., Nature 409,363-366 (2001) ; Elbashir, S. M. , et al., Genes Dev. 15, 188-200 (2001); W00129058 ; W09932619 ; Elbashir SM, et al., 2001 Nature 411: 494-498).

Nucleic acid modulators are commonly used as research reagents, diagnostics, and therapeutics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used to elucidate the function of particular genes (see, for example, U. S. Pat. No. 6,165, 790). Nucleic acid modulators are also used, for example, to distinguish between functions of various members of a biological pathway.

For example, antisense oligomers have been employed as therapeutic moieties in the treatment of disease states in animals and man and have been demonstrated in numerous clinical trials to be safe and effective (Milligan JF, et al, Current Concepts in Antisense Drug Design, J Med Chem. (1993) 36: 1923-1937; Tonkinson JL et al., Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents, Cancer Invest. (1996) 14: 54-65).

Accordingly, in one aspect of the invention, an MCHK-specific nucleic acid modulator is used in an assay to further elucidate the role of the MCHK in the Chkl pathway, and/or its relationship to other members of the pathway. In another aspect of the invention, an MCHK-specific antisense oligomer is used as a therapeutic agent for treatment of Chkl- related disease states.

Assay Systems The invention provides assay systems and screening methods for identifying specific modulators of MCHK activity. As used herein, an"assay system"encompasses all the components required for performing and analyzing results of an assay that detects and/or measures a particular event. In general, primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the MCHK nucleic acid or protein. In general, secondary assays further assess the activity of a MCHK modulating agent identified by a primary assay and may confirm that the modulating agent affects MCHK in a manner relevant to the Chkl pathway. In some cases, MCHK modulators will be directly tested in a secondary assay.

In a preferred embodiment, the screening method comprises contacting a suitable assay system comprising an MCHK polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity (e. g. binding activity), which is based on the particular molecular event the screening method detects. A statistically significant difference between the agent- biased activity and the reference activity indicates that the candidate agent modulates MCHK activity, and hence the Chkl pathway. The MCHK polypeptide or nucleic acid used in the assay may comprise any of the nucleic acids or polypeptides described above.

Primary Assays The type of modulator tested generally determines the type of primary assay.

Primary assaysfor sinall niolecule inodulators For small molecule modulators, screening assays are used to identify candidate modulators. Screening assays may be cell-based or may use a cell-free system that recreates or retains the relevant biochemical reaction of the target protein (reviewed in Sittampalam GS et al., Curr Opin Chem Biol (1997) 1: 384-91 and accompanying references). As used herein the term"cell-based"refers to assays using live cells, dead cells, or a particular cellular fraction, such as a membrane, endoplasmic reticulum, or mitochondrial fraction. The term"cell free"encompasses assays using substantially purified protein (either endogenous or recombinantly produced), partially purified or crude cellular extracts. Screening assays may detect a variety of molecular events, including protein-DNA interactions, protein-protein interactions (e. g. , receptor-ligand binding), transcriptional activity (e. g. , using a reporter gene), enzymatic activity (e. g. , via a property of the substrate), activity of second messengers, immunogenicty and changes in cellular morphology or other cellular characteristics. Appropriate screening assays may use a wide range of detection methods including fluorescent, radioactive, colorimetric, spectrophotometric, and amperometric methods, to provide a read-out for the particular molecular event detected.

Cell-based screening assays usually require systems for recombinant expression of MCHK and any auxiliary proteins demanded by the particular assay. Appropriate methods for generating recombinant proteins produce sufficient quantities of proteins that retain their relevant biological activities and are of sufficient purity to optimize activity and assure assay reproducibility. Yeast two-hybrid and variant screens, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidation of protein complexes. In certain applications, when MCHK-interacting proteins are used in screens to identify small molecule modulators, the binding specificity of the interacting protein to the MCHK protein may be assayed by various known methods such as substrate processing (e. g. ability of the candidate MCHK-specific binding agents to function as negative effectors in MCHK-expressing cells), binding equilibrium constants (usually at least about 107 nul, preferably at least about 108 M-1, more preferably at least about 109 M-1), and immunogenicity (e. g. ability to elicit MCHK specific antibody

in a heterologous host such as a mouse, rat, goat or rabbit). For enzymes and receptors, binding may be assayed by, respectively, substrate and ligand processing.

The screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of a MCHK polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein. The MCHK polypeptide can be full length or a fragment thereof that retains functional MCHK activity. The MCHK polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag. The MCHK polypeptide is preferably human MCHK, or is an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects candidate agent-based modulation of MCHK interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has MCHK-specific binding activity, and can be used to assess normal MCHK gene function.

Suitable assay formats that may be adapted to screen for MCHK modulators are known in the art. Preferred screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening compound libraries for lead compounds (Fernandes PB, Curr Opin Chem Biol (1998) 2: 597-603; Sundberg SA, Curr Opin Biotechnol 2000,11 : 47-53). In one preferred embodiment, screening assays uses fluorescence technologies, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer. These systems offer means to monitor protein-protein or DNA-protein interactions in which the intensity of the signal emitted from dye-labeled molecules depends upon their interactions with partner molecules (e. g. , Selvin PR, Nat Struct Biol (2000) 7: 730-4; Fernandes PB, supra ; Hertzberg RP and Pope AJ, Curr Opin Chem Biol (2000) 4: 445-451).

A variety of suitable assay systems may be used to identify candidate MCHK and Chkl pathway modulators (e. g. U. S. Pat. No. 6,165, 992 (kinase assays); U. S. Pat. Nos.

5,550, 019 and 6,133, 437 (apoptosis assays); U. S. Pat. No. 6,114, 132 (phosphatase and protease assays), U. S. Pat. Nos. 5,976, 782,6, 225,118 and 6,444, 434 (angiogenesis assays), among others). Specific preferred assays are described in more detail below.

Protein phosophatases catalyze the removal of a gamma phosphate from a serine, threonine or tyrosine residue in a protein substrate. Since phosphatases act in opposition to kinases, appropriate assays measure the same parameters as kinase assays. In one example, the dephosphorylation of a fluorescently labeled peptide substrate allows trypsin cleavage of the substrate, which in turn renders the cleaved substrate significantly more fluorescent (Nishikata M et al., Biochem J (1999) 343: 35-391). In another example,

fluorescence polarization (FP), a solution-based, homogeneous technique requiring no immobilization or separation of reaction components, is used to develop high throughput screening (HTS) assays for protein phosphatases. This assay uses direct binding of the phosphatase with the target, and increasing concentrations of target-phosphatase increase the rate of dephosphorylation, leading to a change in polarization (Parker GJ et al. , (2000) J Biomol Screen 5: 77-88).

RNA folds into a myriad of tertiary structures that are responsible for its diverse functions in cells. In most instances, RNA is associated with RNA-binding proteins (RBPs) that protect, stabilize, package or transport RNA, mediate RNA interactions with other biomolecules or act catalytically on RNA. The structural information obtained for RNA alone and RNA-protein complexes has elucidated a variety of RNA tertiary structures and diverse modes for RNA-protein interaction. The specific interaction of proteins with highly structured RNAs makes it possible to target unique RNA motifs with small molecules, thus making RNA an interesting target for therapeutic intervention.

Assays for RNA binding or processing may be based on homogeneous scintillation proximity (Liu J, et al. , Anal Biochem 2001 289: 239-245), chemiluminescense (Mazumder A, Nucleic Acids Res 1998 26: 1996-2000), gel shift (Stull RA, et al., Antisense Nucleic Acid Drug Dev 1996 6: 221-228; U. S. Pat. No: 6004749).

Adapter proteins are involved in a wide range of signaling and other cellular processes and generally facilitate protein-protein or protein-nucleic acid interactions via certain conserved motifs, including PDZ, SH2, SH3, PH, TRAF, WD40, LIM, ankyrin repeat, KH and annexin domains, etc. Assays for adapter protein activity may measure protein binding at the conserved motifs. For instance, exemplary assays for SH2 domain- containing proteins have measured binding using fluorescently labeled peptide substrate and fluorescence polarization or laser-scanning techniques (Lynch BA et al. , Anal Biochem 1999,275 : 62-73 ; Zuck P et al. , Proc Natl Acad Sci USA 1999,96 : 11122- 11127). An alternative SH2 binding assay uses radiolabeled peptide. An assay for protein-protein interaction at the LIM domain has used fluorescently labeled LIM- containing proteins (FHL2 and FHL3) and the fluorescence resonance energy transfer (FRET) technique (Li HY, J Cell Biochem 2001,80 : 293-303).

Transcription factors control gene transcription. Electrophoretic mobility shift assay (EMSA) or gel shift assay is one of the most powerful methods for studying protein- DNA interactions. High throughput gel shift assays for transcription factors may involve fluorescence (Cyano dye Cy5) labeled oligodeoxynucleotide duplexes as specific probes

and an automatic DNA sequencer for analysis (Ruscher K, et al. , (2000) J Biotechnol 78: 163-70). Alternatively high throughput methods involve colorimetric assays (Renard P, et al. (2001) Nucleic Acids Res 29 (4): E21), or homogeneous fluorescence assays for the detection and quantification of sequence-specific DNA-binding proteins (Heyduk T, and Heyduk E (2001) Nat Biotechnol 20: 171-6. ) Assays ATPase activity, such as described in Blackburn et al (Blackburn CL, et al., (1999) J Org Chem 64: 5565-5570), are known in the art. The ATPase assay is performed using the EnzCheck ATPase kit (Molecular Probes). The assays are performed using an Ultraspec spectrophotometer (Pharmacia), and the progress of the reaction are monitored by absorbance increase at 360 nm. Microtubules (1.7 mM final), kinesin (0.11 mM final), inhibitor (or DMSO blank at 5% final), and the EnzCheck components (purine nucleotide phosphorylase and MESG substrate) are premixed in the cuvette in a reaction buffer (40 mM PIPES pH 6.8, 5 mM paclitaxel, 1 mM EGTA, 5 mM MgCl2). The reaction is initiated by addition of MgATP (1 mM final).

Kinase assays. In some preferred embodiments the screening assay detects the ability of the test agent to modulate the kinase activity of an MCHK polypeptide. In further embodiments, a cell-free kinase assay system is used to identify a candidate Chkl modulating agent, and a secondary, cell-based assay, such as an apoptosis or hypoxic induction assay (described below), may be used to further characterize the candidate Chkl modulating agent. Many different assays for kinases have been reported in the literature and are well known to those skilled in the art (e. g. U. S. Pat. No. 6,165, 992; Zhu et al., Nature Genetics (2000) 26: 283-289; and W00073469). Radioassays, which monitor the transfer of a gamma phosphate are frequently used. For instance, a scintillation assay for p56 (lck) kinase activity monitors the transfer of the gamma phosphate from gamma ATP to a biotinylated peptide substrate; the substrate is captured on a streptavidin coated bead that transmits the signal (Beveridge M et al., J Biomol Screen (2000) 5: 205-212).

This assay uses the scintillation proximity assay (SPA), in which only radio-ligand bound to receptors tethered to the surface of an SPA bead are detected by the scintillant immobilized within it, allowing binding to be measured without separation of bound from free ligand.

Other assays for protein kinase activity may use antibodies that specifically recognize phosphorylated substrates. For instance, the kinase receptor activation (KIRA) assay measures receptor tyrosine kinase activity by ligand stimulating the intact receptor

in cultured cells, then capturing solubilized receptor with specific antibodies and quantifying phosphorylation via phosphotyrosine ELISA (Sadick MD, Dev Biol Stand (1999) 97: 121-133).

Another example of antibody based assays for protein kinase activity is TRF (time- resolved fluorometry). This method utilizes europium chelate-labeled anti- phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time- resolved, dissociation-enhanced fluorescence (Braunwalder AF, et al., Anal Biochem 1996 Jul 1 ; 238 (2): 159-64).

Apoptosis assays. Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay. The TUNEL assay is used to measure nuclear DNA fragmentation characteristic of apoptosis (Lazebnik et al., 1994, Nature 371,346), by following the incorporation of fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169,1747). Apoptosis may further be assayed by acridine orange staining of tissue culture cells (Lucas, R. , et al. , 1998, Blood 15: 4730-41). An apoptosis assay system may comprise a cell that expresses an MCHK, and that optionally has defective Chkl function (e. g. Chkl is over-expressed or under- expressed relative to wild-type cells). A test agent can be added to the apoptosis assay system and changes in induction of apoptosis relative to controls where no test agent is added, identify candidate Chkl modulating agents. In some embodiments of the invention, an apoptosis assay may be used as a secondary assay to test a candidate Chkl modulating agents that is initially identified using a cell-free assay system. An apoptosis assay may also be used to test whether MCHK function plays a direct role in apoptosis.

For example, an apoptosis assay may be performed on cells that over-or under-express MCHK relative to wild type cells. Differences in apoptotic response compared to wild type cells suggests that the MCHK plays a direct role in the apoptotic response. Apoptosis assays are described further in US Pat. No. 6,133, 437.

Cell proliferation and cell cycle assays. Cell proliferation may be assayed via bromodeoxyuridine (BRDU) incorporation. This assay identifies a cell population undergoing DNA synthesis by incorporation of BRDU into newly-synthesized DNA.

Newly-synthesized DNA may then be detected using an anti-BRDU antibody (Hoshino et

al., 1986, Int. J. Cancer 38,369 ; Campana et al., 1988, J. Immunol. Meth. 107,79), or by other means.

Cell proliferation is also assayed via phospho-histone H3 staining, which identifies a cell population undergoing mitosis by phosphorylation of histone H3. Phosphorylation of histone H3 at serine 10 is detected using an antibody specfic to the phosphorylated form of the serine 10 residue of histone H3. (Chadlee, D. N. 1995, J. Biol. Chem 270: 20098- 105). Cell Proliferation may also be examined using [3H]-thymidine incorporation (Chen, J. , 1996, Oncogene 13: 1395-403; Jeoung, J. , 1995, J. Biol. Chem. 270: 18367-73). This assay allows for quantitative characterization of S-phase DNA syntheses. In this assay, cells synthesizing DNA will incorporate [H]-thymidine into newly synthesized DNA.

Incorporation can then be measured by standard techniques such as by counting of radioisotope in a scintillation counter (e. g. , Beckman LS 3800 Liquid Scintillation Counter). Another proliferation assay uses the dye Alamar Blue (available from Biosource International), which fluoresces when reduced in living cells and provides an indirect measurement of cell number (Voytik-Harbin SL et al. , 1998, In Vitro Cell Dev Biol Anim 34: 239-46).

Cell proliferation may also be assayed by colony formation in soft agar (Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989) ). For example, cells transformed with MCHK are seeded in soft agar plates, and colonies are measured and counted after two weeks incubation.

Involvement of a gene in the cell cycle may be assayed by flow cytometry (Gray JW et al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med 49: 237-55). Cells transfected with an MCHK may be stained with propidium iodide and evaluated in a flow cytometer (available from Becton Dickinson), which indicates accumulation of cells in different stages of the cell cycle.

Accordingly, a cell proliferation or cell cycle assay system may comprise a cell that expresses an MCHK, and that optionally has defective Chkl function (e. g. Chkl is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the assay system and changes in cell proliferation or cell cycle relative to controls where no test agent is added, identify candidate Chkl modulating agents. In some embodiments of the invention, the cell proliferation or cell cycle assay may be used as a secondary assay to test a candidate Chkl modulating agents that is initially identified using another assay system such as a cell-free assay system. A cell proliferation assay may also be used to test whether MCHK function plays a direct role in cell proliferation or cell cycle. For

example, a cell proliferation or cell cycle assay may be performed on cells that over-or under-express MCHK relative to wild type cells. Differences in proliferation or cell cycle compared to wild type cells suggests that the MCHK plays a direct role in cell proliferation or cell cycle.

Angiogenesis. Angiogenesis may be assayed using various human endothelial cell systems, such as umbilical vein, coronary artery, or dermal cells. Suitable assays include Alamar Blue based assays (available from Biosource International) to measure proliferation; migration assays using fluorescent molecules, such as the use of Becton Dickinson Falcon HTS FluoroBlock cell culture inserts to measure migration of cells through membranes in presence or absence of angiogenesis enhancer or suppressors; and tubule formation assays based on the formation of tubular structures by endothelial cells on Matrigel0 (Becton Dickinson). Accordingly, an angiogenesis assay system may comprise a cell that expresses an MCHK, and that optionally has defective Chkl function (e. g. Chkl is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the angiogenesis assay system and changes in angiogenesis relative to controls where no test agent is added, identify candidate Chkl modulating agents. In some embodiments of the invention, the angiogenesis assay may be used as a secondary assay to test a candidate Chkl modulating agents that is initially identified using another assay system. An angiogenesis assay may also be used to test whether MCHK function plays a direct role in cell proliferation. For example, an angiogenesis assay may be performed on cells that over-or under-express MCHK relative to wild type cells.

Differences in angiogenesis compared to wild type cells suggests that the MCHK plays a direct role in angiogenesis. U. S. Pat. Nos. 5,976, 782,6, 225, 118 and 6,444, 434, among others, describe various angiogenesis assays.

Hypoxic induction. The alpha subunit of the transcription factor, hypoxia inducible factor-1 (HIF-1), is upregulated in tumor cells following exposure to hypoxia in vitro. Under hypoxic conditions, HIF-1 stimulates the expression of genes known to be important in tumour cell survival, such as those encoding glyolytic enzymes and VEGF.

Induction of such genes by hypoxic conditions may be assayed by growing cells transfected with MCHK in hypoxic conditions (such as with 0. 1% 02, 5% C02, and balance N2, generated in a Napco 7001 incubator (Precision Scientific) ) and normoxic conditions, followed by assessment of gene activity or expression by Taqman0. For

example, a hypoxic induction assay system may comprise a cell that expresses an MCHK, and that optionally has defective Chkl function (e. g. Chkl is over-expressed or under- expressed relative to wild-type cells). A test agent can be added to the hypoxic induction assay system and changes in hypoxic response relative to controls where no test agent is added, identify candidate Chkl modulating agents. In some embodiments of the invention, the hypoxic induction assay may be used as a secondary assay to test a candidate Chkl modulating agents that is initially identified using another assay system.

A hypoxic induction assay may also be used to test whether MCHK function plays a direct role in the hypoxic response. For example, a hypoxic induction assay may be performed on cells that over-or under-express MCHK relative to wild type cells. Differences in hypoxic response compared to wild type cells suggests that the MCHK plays a direct role in hypoxic induction.

Cell adhesion. Cell adhesion assays measure adhesion of cells to purified adhesion proteins, or adhesion of cells to each other, in presence or absence of candidate modulating agents. Cell-protein adhesion assays measure the ability of agents to modulate the adhesion of cells to purified proteins. For example, recombinant proteins are produced, diluted to 2. 5g/mL in PBS, and used to coat the wells of a microtiter plate. The wells used for negative control are not coated. Coated wells are then washed, blocked with 1% BSA, and washed again. Compounds are diluted to 2x final test concentration and added to the blocked, coated wells. Cells are then added to the wells, and the unbound cells are washed off. Retained cells are labeled directly on the plate by adding a membrane-permeable fluorescent dye, such as calcein-AM, and the signal is quantified in a fluorescent microplate reader.

Cell-cell adhesion assays measure the ability of agents to modulate binding of cell adhesion proteins with their native ligands. These assays use cells that naturally or recombinantly express the adhesion protein of choice. In an exemplary assay, cells expressing the cell adhesion protein are plated in wells of a multiwell plate. Cells expressing the ligand are labeled with a membrane-permeable fluorescent dye, such as BCECF, and allowed to adhere to the monolayers in the presence of candidate agents.

Unbound cells are washed off, and bound cells are detected using a fluorescence plate reader.

High-throughput cell adhesion assays have also been described. In one such assay, small molecule ligands and peptides are bound to the surface of microscope slides using a

microarray spotter, intact cells are then contacted with the slides, and unbound cells are washed off. In this assay, not only the binding specificity of the peptides and modulators against cell lines are determined, but also the functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip is measured (Falsey JR et al. , Bioconjug Chem. 2001 May-Jun; 12 (3): 346-53).

Tubulogenesis. Tubulogenesis assays monitor the ability of cultured cells, generally endothelial cells, to form tubular structures on a matrix substrate, which generally simulates the environment of the extracellular matrix. Exemplary substrates include Matrigel (Becton Dickinson), an extract of basement membrane proteins containing laminin, collagen IV, and heparin sulfate proteoglycan, which is liquid at 4° C and forms a solid gel at 37° C. Other suitable matrices comprise extracellular components such as collagen, fibronectin, and/or fibrin. Cells are stimulated with a pro-angiogenic stimulant, and their ability to form tubules is detected by imaging. Tubules can generally be detected after an overnight incubation with stimuli, but longer or shorter time frames may also be used. Tube formation assays are well known in the art (e. g. , Jones MK et al., 1999, Nature Medicine 5: 1418-1423). These assays have traditionally involved stimulation with serum or with the growth factors FGF or VEGF. Serum represents an undefined source of growth factors. In a preferred embodiment, the assay is performed with cells cultured in serum free medium, in order to control which process or pathway a candidate agent modulates. Moreover, we have found that different target genes respond differently to stimulation with different pro-angiogenic agents, including inflammatory angiogenic factors such as TNF-alpa. Thus, in a further preferred embodiment, a tubulogenesis assay system comprises testing an MCHK's response to a variety of factors, such as FGF, VEGF, phorbol myristate acetate (PMA), TNF-alpha, ephrin, etc.

Cell Migration. An invasion/migration assay (also called a migration assay) tests the ability of cells to overcome a physical barrier and to migrate towards pro-angiogenic signals. Migration assays are known in the art (e. g. , Paik JH et al. , 2001, J Biol Chem 276: 11830-11837). In a typical experimental set-up, cultured endothelial cells are seeded onto a matrix-coated porous lamina, with pore sizes generally smaller than typical cell size. The matrix generally simulates the environment of the extracellular matrix, as described above. The lamina is typically a membrane, such as the transwell polycarbonate membrane (Corning Costar Corporation, Cambridge, MA), and is generally part of an

upper chamber that is in fluid contact with a lower chamber containing pro-angiogenic stimuli. Migration is generally assayed after an overnight incubation with stimuli, but longer or shorter time frames may also be used. Migration is assessed as the number of cells that crossed the lamina, and may be detected by staining cells with hemotoxylin solution (VWR Scientific, South San Francisco, CA), or by any other method for determining cell number. In another exemplary set up, cells are fluorescently labeled and migration is detected using fluorescent readings, for instance using the Falcon HTS FluoroBlok (Becton Dickinson). While some migration is observed in the absence of stimulus, migration is greatly increased in response to pro-angiogenic factors. As described above, a preferred assay system for migration/invasion assays comprises testing an MCHK's response to a variety of pro-angiogenic factors, including tumor angiogenic and inflammatory angiogenic agents, and culturing the cells in serum free medium.

Sprouting assay. A sprouting assay is a three-dimensional in vitro angiogenesis assay that uses a cell-number defined spheroid aggregation of endothelial cells ("spheroid"), embedded in a collagen gel-based matrix. The spheroid can serve as a starting point for the sprouting of capillary-like structures by invasion into the extracellular matrix (termed"cell sprouting") and the subsequent formation of complex anastomosing networks (Korff and Augustin, 1999, J Cell Sci 112: 3249-58). In an exemplary experimental set-up, spheroids are prepared by pipetting 400 human umbilical vein endothelial cells into individual wells of a nonadhesive 96-well plates to allow overnight spheroidal aggregation (Korff and Augustin: J Cell Biol 143: 1341-52, 1998).

Spheroids are harvested and seeded in 900AI of methocel-collagen solution and pipetted into individual wells of a 24 well plate to allow collagen gel polymerization. Test agents are added after 30 min by pipetting 100 lit of 10-fold concentrated working dilution of the test substances on top of the gel. Plates are incubated at 37°C for 24h. Dishes are fixed at the end of the experimental incubation period by addition of paraformaldehyde. Sprouting intensity of endothelial cells can be quantitated by an automated image analysis system to determine the cumulative sprout length per spheroid.

Primary assays for antibody modulators For antibody modulators, appropriate primary assays test is a binding assay that tests the antibody's affinity to and specificity for the MCHK protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999,

supra). The enzyme-linked immunosorbant assay (ELISA) is a preferred method for detecting MCHK-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.

In some cases, screening assays described for small molecule modulators may also be used to test antibody modulators.

Primary assays for nucleic acid modulators For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit or enhance MCHK gene expression, preferably mRNA expression. In general, expression analysis comprises comparing MCHK expression in like populations of cells (e. g. , two pools of cells that endogenously or recombinantly express MCHK) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art. For instance, Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR (e. g. , using the TaqMan@, PE Applied Biosystems), or microarray analysis may be used to confirm that MCHK mRNA expression is reduced in cells treated with the nucleic acid modulator (e. g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al., Biotechniques (1999) 26: 112-125; Kallioniemi OP, Ann Med 2001,33 : 142-147; Blohm DH and Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12: 41-47). Protein expression may also be monitored. Proteins are most commonly detected with specific antibodies or antisera directed against either the MCHK protein or specific peptides. A variety of means including Western blotting, ELISA, or in situ detection, are available (Harlow E and Lane D, 1988 and 1999, supra).

In some cases, screening assays described for small molecule modulators, particularly in assay systems that involve MCHK mRNA expression, may also be used to test nucleic acid modulators.

Secondary Assays Secondary assays may be used to further assess the activity of MCHK-modulating agent identified by any of the above methods to confirm that the modulating agent affects MCHK in a manner relevant to the Chkl pathway. As used herein, MCHK-modulating agents encompass candidate clinical compounds or other agents derived from previously identified modulating agent. Secondary assays can also be used to test the activity of a

modulating agent on a particular genetic or biochemical pathway or to test the specificity of the modulating agent's interaction with MCHK.

Secondary assays generally compare like populations of cells or animals (e. g. , two pools of cells or animals that endogenously or recombinantly express MCHK) in the presence and absence of the candidate modulator. In general, such assays test whether treatment of cells or animals with a candidate MCHK-modulating agent results in changes in the Chkl pathway in comparison to untreated (or mock-or placebo-treated) cells or animals. Certain assays use"sensitized genetic backgrounds", which, as used herein, describe cells or animals engineered for altered expression of genes in the Chkl or interacting pathways.

Cell-based assays Cell based assays may detect endogenous Chkl pathway activity or may rely on recombinant expression of Chkl pathway components. Any of the aforementioned assays may be used in this cell-based format. Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.

Animal Assays A variety of non-human animal models of normal or defective Chkl pathway may be used to test candidate MCHK modulators. Models for defective Chkl pathway typically use genetically modified animals that have been engineered to mis-express (e. g., over-express or lack expression in) genes involved in the Chkl pathway. Assays generally require systemic delivery of the candidate modulators, such as by oral administration, injection, etc.

In a preferred embodiment, Chkl pathway activity is assessed by monitoring neovascularization and angiogenesis. Animal models with defective and normal Chkl are used to test the candidate modulator's affect on MCHK in Matrigel@ assays. Matrigel0 is an extract of basement membrane proteins, and is composed primarily of laminin, collagen IV, and heparin sulfate proteoglycan. It is provided as a sterile liquid at 4° C, but rapidly forms a solid gel at 37° C. Liquid Matrigel@ is mixed with various angiogenic agents, such as bFGF and VEGF, or with human tumor cells which over-express the MCHK. The mixture is then injected subcutaneously (SC) into female athymic nude mice (Taconic, Germantown, NY) to support an intense vascular response. Mice with MatrigelG) pellets may be dosed via oral (PO), intraperitoneal (IP), or intravenous (IV) routes with the

candidate modulator. Mice are euthanized 5-12 days post-injection, and the Matrigel0 pellet is harvested for hemoglobin analysis (Sigma plasma hemoglobin kit). Hemoglobin content of the gel is found to correlate the degree of neovascularization in the gel.

In another preferred embodiment, the effect of the candidate modulator on MCHK is assessed via tumorigenicity assays. Tumor xenograft assays are known in the art (see, e. g. , Ogawa K et al. , 2000, Oncogene 19: 6043-6052). Xenografts are typically implanted SC into female athymic mice, 6-7 week old, as single cell suspensions either from a pre- existing tumor or from in vitro culture. The tumors which express the MCHK endogenously are injected in the flank, 1 x 105 to 1 x 107 cells per mouse in a volume of 100 pL using a 27gauge needle. Mice are then ear tagged and tumors are measured twice weekly. Candidate modulator treatment is initiated on the day the mean tumor weight reaches 100 mg. Candidate modulator is delivered IV, SC, IP, or PO by bolus administration. Depending upon the pharmacokinetics of each unique candidate modulator, dosing can be performed multiple times per day. The tumor weight is assessed by measuring perpendicular diameters with a caliper and calculated by multiplying the measurements of diameters in two dimensions. At the end of the experiment, the excised tumors maybe utilized for biomarker identification or further analyses. For immunohistochemistry staining, xenograft tumors are fixed in 4% paraformaldehyde, 0. 1M phosphate, pH 7.2, for 6 hours at 4°C, immersed in 30% sucrose in PBS, and rapidly frozen in isopentane cooled with liquid nitrogen.

In another preferred embodiment, tumorogenicity is monitored using a hollow fiber assay, which is described in U. S. Pat No. US 5,698, 413. Briefly, the method comprises implanting into a laboratory animal a biocompatible, semi-permeable encapsulation device containing target cells, treating the laboratory animal with a candidate modulating agent, and evaluating the target cells for reaction to the candidate modulator. Implanted cells are generally human cells from a pre-existing tumor or a tumor cell line. After an appropriate period of time, generally around six days, the implanted samples are harvested for evaluation of the candidate modulator. Tumorogenicity and modulator efficacy may be evaluated by assaying the quantity of viable cells present in the macrocapsule, which can be determined by tests known in the art, for example, MTT dye conversion assay, neutral red dye uptake, trypan blue staining, viable cell counts, the number of colonies formed in soft agar, the capacity of the cells to recover and replicate in vitro, etc.

In another preferred embodiment, a tumorogenicity assay use a transgenic animal, usually a mouse, carrying a dominant oncogene or tumor suppressor gene knockout under

the control of tissue specific regulatory sequences; these assays are generally referred to as transgenic tumor assays. In a preferred application, tumor development in the transgenic model is well characterized or is controlled. In an exemplary model, the"RIPl-Tag2" transgene, comprising the SV40 large T-antigen oncogene under control of the insulin gene regulatory regions is expressed in pancreatic beta cells and results in islet cell carcinomas (Hanahan D, 1985, Nature 315: 115-122; Parangi S et al, 1996, Proc Natl Acad Sci USA 93: 2002-2007; Bergers G et al, 1999, Science 284: 808-812). An"angiogenic switch, "occurs at approximately five weeks, as normally quiescent capillaries in a subset of hyperproliferative islets become angiogenic. The RIP1-TAG2 mice die by age 14 weeks. Candidate modulators may be administered at a variety of stages, including just prior to the angiogenic switch (e. g. , for a model of tumor prevention), during the growth of small tumors (e. g. , for a model of intervention), or during the growth of large and/or invasive tumors (e. g. , for a model of regression). Tumorogenicity and modulator efficacy can be evaluating life-span extension and/or tumor characteristics, including number of tumors, tumor size, tumor morphology, vessel density, apoptotic index, etc.

Diagnostic and therapeutic uses Specific MCHK-modulating agents are useful in a variety of diagnostic and therapeutic applications where disease or disease prognosis is related to defects in the Chkl pathway, such as angiogenic, apoptotic, or cell proliferation disorders. Accordingly, the invention also provides methods for modulating the Chkl pathway in a cell, preferably a cell pre-determined to have defective or impaired Chkl function (e. g. due to overexpression, underexpression, or misexpression of Chkl, or due to gene mutations), comprising the step of administering an agent to the cell that specifically modulates MCHK activity. Preferably, the modulating agent produces a detectable phenotypic change in the cell indicating that the Chkl function is restored. The phrase"function is restored", and equivalents, as used herein, means that the desired phenotype is achieved, or is brought closer to normal compared to untreated cells. For example, with restored Chkl function, cell proliferation and/or progression through cell cycle may normalize, or be brought closer to normal relative to untreated cells. The invention also provides methods for treating disorders or disease associated with impaired Chkl function by administering a therapeutically effective amount of an MCHK-modulating agent that modulates the Chkl pathway. The invention further provides methods for modulating MCHK function in a cell, preferably a cell pre-determined to have defective or impaired

MCHK function, by administering an MCHK-modulating agent. Additionally, the invention provides a method for treating disorders or disease associated with impaired MCHK function by administering a therapeutically effective amount of an MCHK- modulating agent.

The discovery that MCHK is implicated in Chkl pathway provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders involving defects in the Chkl pathway and for the identification of subjects having a predisposition to such diseases and disorders.

Various expression analysis methods can be used to diagnose whether MCHK expression occurs in a particular sample, including Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR, and microarray analysis. (e. g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al., Biotechniques (1999) 26: 112-125; Kallioniemi OP, Ann Med 2001,33 : 142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001,12 : 41-47).

Tissues having a disease or disorder implicating defective Chkl signaling that express an MCHK, are identified as amenable to treatment with an MCHK modulating agent. In a preferred application, the Chkl defective tissue overexpresses an MCHK relative to normal tissue. For example, a Northern blot analysis of mRNA from tumor and normal cell lines, or from tumor and matching normal tissue samples from the same patient, using full or partial MCHK cDNA sequences as probes, can determine whether particular tumors express or overexpress MCHK. Alternatively, the TaqMan@ is used for quantitative RT- PCR analysis of MCHK expression in cell lines, normal tissues and tumor samples (PE Applied Biosystems).

Various other diagnostic methods may be performed, for example, utilizing reagents such as the MCHK oligonucleotides, and antibodies directed against an MCHK, as described above for: (1) the detection of the presence of MCHK gene mutations, or the detection of either over-or under-expression of MCHK mRNA relative to the non- disorder state; (2) the detection of either an over-or an under-abundance of MCHK gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in the signal transduction pathway mediated by MCHK.

Thus, in a specific embodiment, the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in MCHK expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for MCHK expression; c) comparing results from step (b) with a

control; and d) determining whether step (c) indicates a likelihood of the disease or disorder. Preferably, the disease is cancer. The probe may be either DNA or protein, including an antibody.

EXAMPLES The following experimental section and examples are offered by way of illustration and not by way of limitation.

I. Drosophila Chkl screen The Drosophila Chkl gene was overexpressed specifically in the eye using the GAL4/UAS system (Brand, A. H. & Perrimon, N. Development (1993) 118: 401-415).

The glass multimer repeats enhancer was used to drive expression of the GAL4 transcription factor in the eye (GMR-GAL4). GAL4 activated expression of Drosophila Chkl by initiating transcription from UAS sites contained within a transposon inserted in the first intron of the Chkl gene (UAS-Chkl). Overexpression of Chkl in the eye resulted in a G2 cell cycle arrest and a deterioration of general eye morphology. In a screen to identify suppressors and enhancers of Drosophila Chkl, females carrying one copy each of GMR-GAL4 and UAS-Chkl were crossed to 5300 males carrying random insertions of a piggyBac transposon (Fraser M et al., Virology (1985) 145: 356-361). Progeny containing insertions were compared to non-insertion-bearing sibling progeny for enhancement or suppression of the Chkl phenotype. Sequence information surrounding the piggyBac insertion site was used to identify the modifier genes, which are new members of the Chkl DNA damage response pathway.

II. Analysis of Table 1 BLAST analysis (Altschul et al., supra) was employed to identify orthologs of Drosophila modifiers. The columns"MCHK symbol", and"MCHK name aliases" provide a symbol and the known name abbreviations for the Targets, where available, from Genbank."MCHK RefSeq_NA or GI_NA","MCHK GIAA","MCHK NAME", and"MCHK Description"provide the reference DNA sequences for the MCHKs as available from National Center for Biology Information (NCBI), MCHK protein Genbank identifier number (GI#), MCHK name, and MCHK description, all available from Genbank, respectively. The length of each amino acid is in the"MCHK Protein Length" column.

Names and Protein sequences of Drosophila modifiers of Chkl from screen (Example I), are represented in the"Modifier Name"and"Modifier GIAA"column by GI&num , respectively.

Table 1 MCHK MCHK NAME MCHK NA MCHK AA MCHK MCHK DESCRIPTION MCHK MODIFIER MODIFIER SYMBOL ALIASES RefSeq_NA EQ GI_AA SEQ NAME PROTEIN NAME GI_AA or GI#_NA ID ID LENGTH NO : NO : AWP1 hypothetical NM_019006 1 14753880 51 protein Associated with 208 CG16751 7299163 protein; Xi044547 associated PRKCL1, contains zf- associated with with A20 and zf-ANI zinc PRKI, protein PRK1 finger domains, interacts associated with with serine/threonine PRK1 kinase PRK1 (PRKCLI), may play a role in signal transduction and in developmental processes LOC1477, similar to zinc XM_085861 18589968 52 similar to na 167 CG16751 7299163 24 finger protein zinc finger 216 protein 216 TEX27 FLJ13222, likel NM_021943 3 11345684 53 testis Protein containing two 227 CG16751 7299163 yorthologof XM011394 expressed ANl-like zinc finger mouse testis sequence domains, has weak expressed gene 27 similarity to 27, testis uncharacterized mouse expressed Awpl, which may sequence 27 function in signal transduction ZNF216, zinc finger NM OOG007 5174755 4 zinc finger Protein with homology 213 CG16751 7299163 protein 216 protein to zinc-finger proteins, 216 expressed in the cochlea KIAA117 , KIAA1171 XM_054958 5 6330204 55 KIAA117 Protein of unknown 595 CG9339 17862132 1 protein 1 protein function PTPN9 MEG2, PTPase-NM_002833 4506301 56 protein Protein tyrosine 593 CG3101 18858107 MEG2, protein- tyrosine phosphatase non- tyrosine phosphata receptor type 9, non- phosphatase se, non-membrane spanning MEG2, protein receptor tyrosine phosphatase tyrosine type 9 that may play a role in phosphatase, protein transport non-receptor through the Golgi type 9 apparatus, may be involved in the transport of hydrophobic ligands, may be associated with autism KLF16 DRI2F, BTEB4, NM_031918 13994287 7 Kruppel-Proteinwithhigh 252 EP2237 7296197 BTE-binding like factor similarity to spl-like protein 4, likely 16 zinc finger transcription ortholog of factor (human KLF13), mouse which is a strong dopamine activator of RANTES receptor expression in T cells, regulating contains three C2H2 factor, Kruppel-type zinc finger like factor 16 domains, which bind nucleic acids TIEG EGRA, KLF10, NM_005655 8 5032177 8 TGFB TGFB inducible early 480 EP2237 7296197 TGFB inducible inducible growth response, a early growth early transcription factor with response growth zinc-finger and SH3 response domains; induces apoptosis in pancreatic cells TIEG2 FKLF, KI. FI l, NM 003597 4507503 9 TGFB TGFB inducible early 512 EP2237 7296197 TGFB inducible inducible growth response 2, early growth early binds both DNA and response 2 growth protein, involved in the response 2 regulation of cell growth, activates and inhibits transcription ID1 Inhibitor of NM_002165 10 4504569 60 inhibitor Inhibitor of DNA 154 emc 17647385 DNA binding 1, XM_046179 of DNA binding 1, dominant binding 1, transcriptional negative, dominant corepressor that inhibits inhibitor of negative helix-loop-helix DNA binding 1, helix-transcription factors via dominant loop-helix a dominant-negative negative helix-protein mechanism resulting in loop-helix increased cell growth, protein inhibition of cellular differentiation, and oncogenic cell characteristics ID2 Inhibitor of NM_002166 11 45047571 61 inhibitor Member of the Id helix-134 emc 17647385 DNA binding 2, XMJM5365 of DNA loop-helix family of dominant binding 2, proteins that may negative, dominant negatively regulate cell inhibitor of negative differentiation by DNA binding 2, helix-inhibiting helix-loop- dominant loop-helix helix transcription negative helix-protein factors via a dominant- loop-helix negative mechanism protein ID3 HEIR- NM_002167 12 14726905 62 inhibitor inhibitor of DNA 119 emc 17647385 l, Inhibitor of XM_031306 of DNA binding 3, basic helix- DNA binding 3, binding 3, loop-helix family dominant dominant member that negatively negative, helix-negative regulates cell loop-helix, helix-differentiation by inhibitor of loop-helix inhibiting DNA binding DNA binding 3, protein of certain bHLH dominant transcription factors; negative helix-corresponding gene is loop-helix mutated in protein neuroblastoma and seminoma ID4 Inhibitor of NM_001546 13 4504573 3 inhibitor Inhibitor of DNA-161 emc 17647385 DNA binding 4, M004283 of DNA binding 4, has a HLH- dominant binding 4, dimerization domain but negative helix-dominant lacks a DNA-binding loop-helix, negative domain, inhibits inhibitor of helix-transcription activation DNA binding 4, loop-helix by HLH factors by dominant protein forming heterodimers, negative helix-may be involved in loop-helix pathogenesis of breast, protein ovarian cancer by inhibiting BRCA1 expression ATP1A1 ATPase, NM_000701 14 4502269 4 ATPase, Alpha 1 subunit of the 681 Atpalpha 18487811 Na+K+ Na+/K+ Na+-K+-transporting transporting, transporti ATPase, required for alpha-1 ng, alpha active transport of polypeptide, I sodium in the kidney ATPase, polypeptid and other tissues, may Na+/K+ e be involved in blood transporting, pressure regulation; alpha 1 genetic interaction with polypeptide the NKCC2 gene may cause essential hypertension ATP1A2 ATPase, NMJM0702 15 4502271 65 ATPase, Alpha 2 subunit of the 1020 Atpalpha 18487811 Na+K+ Na+/K+ Na+-K+-transporting transporting, transporti ATPase, required for alpha-2 ng, alpha maintenance of Na+ and polypeptide, 2 (+) K+ gradients across the ATPase, polypeptid plasma membrane; Na+/K+ e genetic variants may be transporting, associated with alpha 2 (+) susceptibility to polypeptide increased blood pressure in response to lead exposure ATP1A3, ATPase, NM_000703 16 4502273 66 ATPase, Alpha 3 subunit of 1013 Atpalpha 18487811 Na+/K+ Na+/K+ Na+/K+ ATPase, transporting, transporti sensitive to inhibition by alpha 3 ng, alpha ouabain polypeptide 3 polypeptid _ e LHX2 LH2, LH- NM004789 17 4758674 67 LIM LIM homeobox protein 389 ap 17647159 2, hLhx2, LIM XM_044817 homeobox 2, transcription factor, HOX gene 2, protein 2 may play a role in cell UM homeobox growth and protein 2 maintenence, highly expressed in chronic myelogenous leukemias LHX9 LIM-homeobox XM 097483 18 12643346 68 LIM LIM homeobox 9, a 388 ap 17647159 9, LIM homeobox member of the homeobox 9 9 LIM/homeodomain- containing family of transcription factors PUM1 PUMH, PUMH1 NM_014676 19 13491166 69 pumilio |Protein containing 1186 pum 7299139 , PUML1, KIAA XM055629 homolog 1 Pumilio-family RNA 0099,1 (Drosophi binding domains, has KIAA0099, PU la) moderate similarity to ML1- D. melanogaster PENDING, pum Pumilio (Pum), which is ilio a maternally expressed (Drosophila) gene required for homolog 1, development of pumilio abdominal segments, homolog 1 contains Pumilio-family (Drosophila) RNA binding domains PUM2 PUMH2, PUML NM_015317 20 13491168 70 pumilio Protein containing eight 1064 pum 7299139 2, KIAA0235, pu homolog 2 Pumilio-family (Puf) milio (Drosophi domain s, which bind (Drosophila) la) RNA, has a region of homolog 2, low similarity to a pumilio region of S. cerevisiae homolog 2 Mpt5p, which is (Drosophila) required for high temperature growth, for recovery from alpha- factor arrest, and for normal lifespan QKI QK, homolog 7542352 21 7542353 71 homolog Quaking, a putative 315 how 17738117 of mouse of mouse RNA-binding protein ; quaking QKI quaking mutations in mouse qk (KH domain QKI (KH lead to effects ranging RNA binding domain from a transient quaking protein) RNA phenotype caused by binding demyelination to protein) embryonic death PPP1R3A PPP1R3, NM 002711 2 450G011 2 protein Protein phosphatase 1 1122 CG9619 7293805 protein XM_054777 phosphata regulatory subunit 3A, phosphatase 1, se 1, may be involved in regulatory regulatory insulin (INS)- (inhibitor) (inhibitor) dependent synthesis of subunit 3A subunit glycogen; mutations are (glycogen and 3A associated with insulin sarcoplasmic glycogen resistance, reticulum and hypersecretion of binding subunit, sarcoplas insulin, type 2 diabetes skeletal muscle) mic and colorectal, lung and reticulum ovarian cancers binding subunit, skeletal muscle) PPP1R3D PPP1R6,protein NM_006242 23 5453948 73 protein Protein phosphatase 1 299 CG9619 7293805 phosphatase 1, XM_009591 phosphata regulatory 6, glycogen regulatory se 1, targeting subunit of subunit 6, regulatory protein phosphatase 1, spinophilin, subunit functions in glycogen protein 3D metabolism in muscle phosphatase 1, regulatory subunit 3D FLJ20257 , hypothetical NM_019606 24 9624990 74 hypothetic Protein of unknown 220 bin3 17647201 protein al protein function FLJ20257 FLJ20257 MGC1125, hypothetical NM032350 25"14150149 75hypothetic Protein of unknown 194 CG15877 7292231 7 protein al protein function, has high MGC11257 MGC1125 similarity to 7 uncharacterized mouse 3110082I17Rik CALM1 CAMI, PHKD, D NM-006888 26 5901912 76 calmoduli Calmodulin 1, member 149 cam 17647231 D132, CALML2 n 1 of the EF-hand domain- , calmodulin 1 (phosphor containing superfamily (phosphorylase ylase of calcium-binding kinase, delta) kinase, proteins, regulates the delta) calcium-dependent activity of proteins and enzymes including ion channels, kinases, phosphatases, and phosphodiesterases CALM2 PHKD, CAMII, NM001743 7 4502549 7 calmoduli Calmodulin 2 149 cam 17647231 phosphorylase XM_057664 n 2 (phosphorylase kinase kinase delta, (phosphor delta), member of the calmodulin 2 ylase calmodulin family of (phosphorylase kinase, calcium binding kinase, delta) delta) proteins involved in calcium dependent signal transduction, expression is largely restricted to the brain CALM3 PHKD, Calmod NM_005184 28 4885109 78 calmoduli Calmodulin 3, putative 152 cam 17647231 ulin-3, XM_028275 n 3 calcium-binding protein calmodulin 3 (phosphor and regulatory delta- (phosphorylase ylase subunit of kinase, delta) kinase, phosphorylase kinase in delta) skeletal muscle LASP1 MLN50,Lasp-1, NM_006148 5453710 79 LIM and LIM and SH3 protein 1, 261 CG3849 7294071 LIM and SH3 XM_008150 SH3 member of a subfamily protein 1 protein 1 of LIM proteins that contains a LIM domain and an SH3 (Src homology region 3) domain, binds actin; gene amplification is detected in breast carcinomas NEB NEM2, nebulin NM_004543 30 4758794 80 nebulin Nebulin, a giant 6669 CG3849 7294071 XM040435 filamentous protein that contains SH3 domains, may function as a molecular ruler to determine thin filament length in skeletal muscles ; genetic variants are associated with autosomal recessive nemaline myopathy NEBL actin-binding Z- NM_006393 31 5453758 81 nebulette Nebulette, binds actin, 1014 CG3849 7294071 disk protein, XM016290 alpha-actinin (ACTN1), nebulette zyxin (ZYX), and tropomyosin, may regulate Z line assembly in cardiac muscle sarcomeres ; polymorphisms in the corresponding gene are associated with nonfamilial idiopathic dilated cardiomyopathy (IDC) CAD1933 17894706 32 17894707 82 na na 349 CG6393 10727027 2 MGC1694 , similar to NM_080663 33 18087835 83 similar to na 328 CG6393 10727027 3 RIKEN cDNA RIKEN 4933424N09 cDNA gene 4933424N 09 gene PRNPIP PINT1, MGC26 XM_046498 4 14742781 84 prion Protein of unknown 337 CG6393 10727027 83, FLJ22943, li protein function kely ortholog of interacting mouse prion protein interactor PINT1, prion protein interacting protein CFL1 CFL, cofilin 1 NM_005507 35 5031635 85 cofilin Non-muscle cofilin, an 166 tsr 17136986 (non-muscle) (non-actin-binding protein muscle) that assists translocation of actin from the cytoplasm to the nucleus; inactivation through phosphorylation by LIM-kinase leads to actin depolymerization CFL2, cofilin 2 NM_021914 36 14719392 86 cofilin 2 Cofilin 2 (muscle 166 tsr 17136986 (muscle) XM_052827 (muscle cofilin), muscle isoform of the cofilin family of actin-binding proteins, may play a role in muscle function and muscle regeneration DSTN ADF, ACTDP, d NM_006870 7 58029G6 87 destrin Actin depolymerizing 165 tsr 17136986 estrin, destrin (actin factor, a member of the (actin depolymer ADF/cofilin family that depolymerizing izing binds to actin and is factor) factor) predicted to depolymerize F-actin by altering filament tilt and twist; present in rod-like inclusions within neurons in the brains of Alzheimer's patients LOC1280 , similar to XM_060798 38 17447226 88 similar to na 170 tsr 17136986 37 cofilin 1, non- cofilin 1, muscle non- muscle LOC1317, similar to XM_067543 39 17446541 89 similar to na 180 tsr 17136986 81 cofilin 1 (non-cofilin 1 muscle) (non- muscle) LOC1594 , similar toXM089606M 1857531690 similar to na 136 tsr 17136986 68 cofilin 1 (non-cofilin 1 muscle) (non- muscle) LOC9056 , similar to XM_032603 41 14784145 91 similar to na 144 tsr 17136986 3 Destrin, Nmr, Destrin, Minimized Nmr, Average Minimize Structure d Average Structure NFKBIL2 IKBR, I-kappa- NM_013432 42 15718772 92 nuclear NF-kappaB inhibitor-1219 CG7457 7295200 B-related factor of like 2, member of protein, nuclear kappa IkappaB family, inhibits factor of kappa light DNA binding of light polypeptid NFKB1-RELA NF- polypeptide e gene kappaB heterodimers gene enhancer enhancer and NFKB1 in B-cells in B-cells homodimers, NF- inhibitor-like 2 inhibitor-kappaB-mediated like 2 transcription from Igkappa enhancer, and may regulate NF- kappaB function in epithelial cells ATP2C1 HHD, BCPM, P NM_014382 43 7656910 3 ATPase, ATPase (Ca2+ 919 CG7651 7296577 MR1, ATP2C1 Ca++ transporting) type 2c A, KIAA1347, transporti member 1, a a Ca2+- ATPase, Ca++ ng, type transporting P-type transporting, 2C, ATPase involved in type 2C, member 1 Ca2+ homeostasis that member 1 may also may play a role in epidermal differentiation ; mutations in the gene cause Hailey-Hailey disease, a blistering skin disease KLAA070, KIAA0703 NM_014861 44 7662256 94 KIAA070 Protein with high 963 CG7651 7296577 3 gene product XMJM7833 3 gene similarity to yeast product Pmrlp, a Ca2+- transporting P-type ATPase of Golgi membrane, member of the El-E2 ATPase family of cation transporters LOC5169 , hHDCforNM016217 457706435 95hHDC for Protein of unknown 543 hdc 18488891 6 homolog of XM_004264 homolog function Drosophila of headcase Drosophil a headcase NCAMI CD56, NCAM, NM_000615 46 10834990 96 neural cell Neural cell adhesion 848 Fas2 7290471 neural cell XM_041415 adhesion molecule 1, a member adhesion molecule of the immunoglobulin molecule 1 1 superfamily, important for cell adhesion between neurons and at neuromuscular junctions, functions to regulate axonal outgrowth and neuronal remodeling NCAM2 neural cell NM_004540 47 4758764 97 neural cell Neural cell adhesion 837 Fas2 7290471 adhesion M009705 adhesion molecule 2, member of molecule 2 molecule the immunoglobulin precursor, 2 superfamily, contains a neural cell transmembrane domain adhesion and fibronectin type m molecule 2 domains in the extracellular domain, may mediate neuronal cell adhesion LOC1627, similar to XM_091768 48 18588797 98 similar to na 295 CG8185 7303060 25 C08B11.4.p C08B11.4 .p PAK3 MRX30,PAK3b NM_002578.49 4505601 99 p21 p21 (CDKN1A)- 544 Pak 17864254 eta, bPAK, hPA I (CDKNI activated kinase 3, K3, CDKNIA, p A) -downstream effector of 21-activated activated members of the rho kinase-3, p21 kinase 3 family of GTPases, such (CDKN1A)- as CDC42 and RACI, activated kinase kinase activity is 3 stimulated by Rac ; mutation of the corresponding gene causes nonsyndromic X- linked mental retardation PAK2 PAK65, PAKga NM_002577. 50 4505599 100 p21 p21 (CDKN1A)-525 Pak 17864254 mma, hPAK65, n 1) XM0393S (CDKN1 activated kinase 2, binds ovel serine 4.7 A)-CDC42 and RACl, kinase, p21 activated activated by caspase (CDKN1A)-kinase 2 cleavage during stress- activated kinase induced apoptosis 2 leading to activation of the c Jun N terminal kinase (JNK) pathway, and may regulate actin organization and smooth muscle contraction

HI. High-Throughput In Vitro Fluorescence Polarization Assay Fluorescently-labeled MCHK peptide/substrate are added to each well of a 96-well microtiter plate, along with a test agent in a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6). Changes in fluorescence polarization, determined by using a Fluorolite FPM-2 Fluorescence Polarization Microtiter System (Dynatech Laboratories, Inc), relative to control values indicates the test compound is a candidate modifier of MCHK activity.

IV. High-Throughput In Vitro Binding Assay.

33P-labeled MCHK peptide is added in an assay buffer (100 mM KC1, 20 mM HEPES pH 7.6, 1 mM MgCl2, 1% glycerol, 0. 5% NP-40,50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors) along with a test agent to the wells of a Neutralite-avidin coated assay plate and incubated at 25°C for 1 hour. Biotinylated substrate is then added to each well and incubated for 1 hour. Reactions are stopped by washing with PBS, and counted in a scintillation counter. Test agents that cause a difference in activity relative to control without test agent are identified as candidate Chkl modulating agents.

V. Immunoprecipitations and Immunoblotting For coprecipitation of transfected proteins, 3 x 106appropriate recombinant cells containing the MCHK proteins are plated on 10-cm dishes and transfected on the following day with expression constructs. The total amount of DNA is kept constant in each transfection by adding empty vector. After 24 h, cells are collected, washed once with phosphate-buffered saline and lysed for 20 min on ice in 1 ml of lysis buffer containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20 mM-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenyl phosphate, 2 mM dithiothreitol, protease

inhibitors (complete, Roche Molecular Biochemicals), and 1 % Nonidet P-40. Cellular debris is removed by centrifugation twice at 15,000 x g for 15 min. The cell lysate is incubated with 25 Itl of M2 beads (Sigma) for 2 h at 4 °C with gentle rocking.

After extensive washing with lysis buffer, proteins bound to the beads are solubilized by boiling in SDS sample buffer, fractionated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluorid membrane and blotted with the indicated antibodies. The reactive bands are visualized with horseradish peroxidase coupled to the appropriate secondary antibodies and the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech).

VI. Kinase assay A purified or partially purified MCHK is diluted in a suitable reaction buffer, e. g., 50 mM Hepes, pH 7.5, containing magnesium chloride or manganese chloride (1-20 mM) and a peptide or polypeptide substrate, such as myelin basic protein or casein (1-10 yg/ml). The final concentration of the kinase is 1-20 nM. The enzyme reaction is conducted in microtiter plates to facilitate optimization of reaction conditions by increasing assay throughput. A 96-well microtiter plate is employed using a final volume 30-100/tu. The reaction is initiated by the addition of 33P-gamma-ATP (0. 5, uCi/ml) and incubated for 0.5 to 3 hours at room temperature. Negative controls are provided by the addition of EDTA, which chelates the divalent cation (Mg2+ or Mon2+) required for enzymatic activity. Following the incubation, the enzyme reaction is quenched using EDTA. Samples of the reaction are transferred to a 96-well glass fiber filter plate (MultiScreen, Millipore). The filters are subsequently washed with phosphate-buffered saline, dilute phosphoric acid (0.5%) or other suitable medium to remove excess radiolabeled ATP. Scintillation cocktail is added to the filter plate and the incorporated radioactivity is quantitated by scintillation counting (Wallac/Perkin Elmer). Activity is defined by the amount of radioactivity detected following subtraction of the negative control reaction value (EDTA quench).

VIt. Expression analysis All cell lines used in the following experiments are NCI (National Cancer Institute) lines, and are available from ATCC (American Type Culture Collection, Manassas, VA 20110-2209). Normal and tumor tissues are obtained from Impath, UC Davis, Clontech, Stratagene, Ardais, Genome Collaborative, and Ambion.

TaqMan analysis is used to assess expression levels of the disclosed genes in various samples.

RNA is extracted from each tissue sample using Qiagen (Valencia, CA) RNeasy kits, following manufacturer's protocols, to a final concentration of 50ng/1lL Single stranded cDNA is then synthesized by reverse transcribing the RNA samples using random hexamers and 500ng of total RNA per reaction, following protocol 4304965 of Applied Biosystems (Foster City, CA).

Primers for expression analysis using TaqMan assay (Applied Biosystems, Foster City, CA) are prepared according to the TaqMan protocols, and the following criteria: a) primer pairs are designed to span introns to eliminate genomic contamination, and b) each primer pair produced only one product. Expression analysis is performed using a 7900HT instrument.

Taqman reactions are carried out following manufacturer's protocols, in 25 ul total volume for 96-well plates and 10 ul total volume for 384-well plates, using 300nM primer and 250 nM probe, and approximately 25ng of cDNA. The standard curve for result analysis is prepared using a universal pool of human cDNA samples, which is a mixture of cDNAs from a wide variety of tissues so that the chance that a target will be present in appreciable amounts is good. The raw data are normalized using 18S rRNA (universally expressed in all tissues and cells).

For each expression analysis, tumor tissue samples are compared with matched normal tissues from the same patient. A gene is considered overexpressed in a tumor when the level of expression of the gene is 2 fold or higher in the tumor compared with its matched normal sample. In cases where normal tissue is not available, a universal pool of cDNA samples is used instead. In these cases, a gene is considered overexpressed in a tumor sample when the difference of expression levels between a tumor sample and the average of all normal samples from the same tissue type is greater than 2 times the standard deviation of all normal samples (i. e., Tumor-average (all normal samples) > 2 x STDEV (all normal samples)).

A modulator identified by an assay described herein can be further validated for therapeutic effect by administration to a tumor in which the gene is overexpressed. A decrease in tumor growth confirms therapeutic utility of the modulator. Prior to treating a patient with the modulator, the likelihood that the patient will respond to treatment can be diagnosed by obtaining a tumor sample from the patient, and assaying for expression of the gene targeted by the modulator. The expression data for the gene (s) can also be used as a diagnostic marker for disease progression. The assay can be performed by expression analysis as described above, by antibody directed to the gene target, or by any other available detection method.