FIELD OF THE INVENTION The invention is, in general, in the field of cell growth regulation. More specifically, the invention provides methods and reagents for detecting cell proliferation, and for modulating cell death and cell proliferation by modulating polo-like kinases.

BACKGROUND OF THE INVENTION The cell cycle is a regulated series of events which culminates in the production of two identical daughter cells from the original, dividing cell, and is characterised by four temporally discrete stages known as Gap 1 (G1), Synthesis (S), and Gap 2 (G2), collectively term "interphase, "and mitosis (M). Non-dividing, for example, terminally differentiated, cells are not considered to be part of the cell cycle. Aberrations in cell cycle processes often result in disorders associated with uncontrolled cellular proliferation, or with diseases associated with cell death or apoptosis. Thus, cancers may be caused by excessive cell growth, out of proportion to cell death, resulting in rapid proliferation. By contrast, excessive cell death, out of proportion to cell regeneration, can result in the destruction of crucial areas of tissue as observed in many degenerative diseases.

The onset of mitosis is marked by extensive morphological reorganisation of cellular structures, accompanied by suppression of the transcription and translation machinery due to, for example, chromosome condensation and nucleolar disassembly. These events result either directly or indirectly from the activation of the cyclin-dependent kinase-1 (CDKl)/cyclin B complex and the concomitant phosphorylation of various substrate proteins (Peter et al. 1990a ; Moreno and Nurse 1990).

B23, also known as nucleophosmin, numatrin, NPM, and N038 (Peter et al. , 1990b), is a multifunctional, highly acidic nucleolar phosphoprotein that shares significant sequence similarity at its N-terminus with the nucleoplasmin family of proteins, and is highly conserved

in vertebrates. B23 was originally identified as a major phosphoprotein localised primarily in the granular and fibrillar regions of the nucleolus (Prestayko et al., 1974 ; Spector et al. , 1984; Yung et al. , 1985).

B23 is associated with preribosomal particles, where it plays a major role in ribosome biogenesis and transport. Its ability to shuttle between the nucleus and the cytoplasm, and to bind to nuclear localisation signal containing proteins has implicated it in nuclear protein trafficking (Borer et al. , 1989). Its abundance has been directly correlated with the proliferation states of cells (Sirri et al. , 1997). Moreover, B23 also acts as a molecular chaperone in preventing protein aggregation and protecting proteins from heat denaturation (Szebeni et al. , 1999). B23 can form hexamers or larger oligomers in vivo, which may be related to its chaperoning activity. Recombinant B23 protein induces sperm chromatin decondensation in vitro through the interaction between its acidic region and core histone proteins (Okuwaki et al. , 2001). Various activities of B23 have been mapped to separate, but partially overlapping domains, of the polypeptide chain (Hingorani et al. , 2000).

Phosphorylation of B23 has been implicated in several different cellular functions including nucleolar disassembly/reassembly (Peter et al. , 1990), ribosome biogenesis (Okuwaki et al. , 2002), centrosome duplication (Okuda et al. ,, 2000; Tokuyama et al,, 2001) and chromosome condensation (Lu et al. , 1996). B23 has been characterised as a CDK1 substrate, both in vitro and in vivo, and its phosphorylation has been implicated in controlling mitotic changes in nucleolar structure and activity (Peter et al. , 1990b). Dephosphorylation of B23 using a general protein kinase inhibitor, staurosporine, induced chromosome decondensation during mitosis (Lu et al. , 1996). Phosphorylation of B23 on threonine residues by cyclin dependent kinases (CDK's) was found to impair its RNA binding activity, consistent with reduced ribosome biogenesis during mitosis (Okuwaki et al. , 2002), and phosphorylation on Thr-199, specifically, by CDK2/cyclin E was shown to be a prerequisite for centrosome duplication (Okuda et al. , 2000, Tokuyama et al. , 2001). While mitotic phosphorylation of B23 occurs primarily on threonine residues, some serine phosphorylation during mitosis has been reported (Jiang et al. , 2000).

Polo-like kinases (Plk's) are protein-serine/threonine kinases that have been implicated in cell cycle control. Plkl, the human homologue of the polo gene product of Drosophila 7nelanogaster, has been implicated in the functional maturation of centrosomes, and for entry into mitosis (Nigg et al. , 1996). Plkl activity is regulated by phosphorylation and cell-cycle related changes in expression levels, and peaks during M phase (Nakajima et al. 2003).

SUMMARY OF THE INVENTION Much effort has been devoted to developing therapeutic and diagnostic agents that affect cell proliferation or survival, for example, cancer drugs that can selectively kill fast proliferating tumour cells while exerting minimal effects on surrounding normal cells. An increased understanding of the molecular basis for eukaryotic cell cycle regulation has led to the search for small molecular inhibitors for CDK's, to inhibit cell cycle progression in tumour cells. Given the correlation between aberrations in the cell cycle and disorders relating to cell proliferation or cell survival, it would be useful to develop new reagents and assays for modulating cell proliferation and survival. Since Plk's are important regulators and targets of CDK's and the cell cycle, they are attractive drug targets. The invention provides, in part, a novel serine phosphorylation site at position 4 on B23, and identifies Plk's as being capable of phosphorylating this site.

In one aspect, the invention provides a method of screening for a compound that modulates the activity of a polo-like kinase, by providing a system including a polo-like kinase or a biologically-active fragment thereof, and a B23 polypeptide that is capable of being phosphorylated by the polo-like kinase, or a fragment, analog, or variant thereof; contacting the system with a test compound; and determining whether the test compound modulates the phosphorylation of the B23 polypeptide, fragment, analog, or variant thereof. The polo-like kinase may be a Plkl, Plk2, Plk3 and Plk4, or a fragment, analog, or variant thereof. In alternative embodiments, the polo-like kinase may be human, for example, human Plkl or a fragment, analog, or variant thereof. In alternative embodiments, the B23 polypeptide may be human, mouse, rat, chicken, and frog B23, or a fragment, analog, or variant thereof. In alternative embodiments, the modulating may be at position 4 of the B23 polypeptide.

In alternative aspects, the invention provides a method of screening for a compound that modulates activity of a B23 polypeptide, or a fragment, analog, or variant thereof, by providing the B23 polypeptide or fragment, analog, or variant thereof under conditions suitable for modulating phosphorylation; providing the test compound; and determining whether the test compound modulates phosphorylation of a B23 or a fragment, analog, or variant thereof at position 4. In some embodiments, the test compound is a kinase, for example, a polo-like kinase. In alternative embodiments, the activity is chromosome decondensation.

In alternative embodiments of the invention, the determining is done using an antibody that specifically binds a B23 polypeptide or fragment, analog, or variant thereof that is

phosphorylated at position 4, for example, the anti-phospho-Mekl/2 antibody. The determining may be done in vivo or in vitro, and the screening may be done using a cell-free system or may be done in a cell.

The invention provides a compound, for example a negative modulator or a positive modulator, determined using any of the methods of the invention, and provides for the use of such compounds for the preparation of a medicament for, for example, treating a cell proliferation disease. The invention also provides a pharmaceutical composition including a compound determined according to the methods of the invention in combination with a physiologically acceptable carrier for, for example, treating a cell proliferation disease.

In alternative aspects, the invention provides a method of screening for a mitotic cell, by providing a test sample; providing a control sample; and detecting a B23 polypeptide that is phosphorylated at Serine-4 in the test sample and the control sample, where an increase in the level of phosphorylation of the B23 polypeptide at Serine-4 in the test sample as compared to the control sample is indicative of a mitotic cell the control sample may include non-cancer cells. The the cell that is undergoing mitosis may be a cancer cell. The detecting may be done using an antibody that specifically binds to the B23 polypeptide that is phosphorylated at Serine-4, for example, the anti-phospho-Mek1/2 antibody. The antibody may be raised against a B23 peptide sequence, and may be detectably labelled. In alternative embodiments, the detecting may be done using a technique selected from the group consisting of ELISA, immunoblotting, and immunoprecipitation, and may be done in vitro or in vivo. The screening may be done using a cell-free system or in a cell.

In alternative aspects, the invention provides a method of treating a cell proliferative disease in a mammal in need of such treatment by administering to the mammal a therapeutically-effective compound that recognises a B23 polypeptide that is phosphorylated at Serine-4. In alternative embodiments, the compound may modulate phosphorylation of a B23 polypeptide at position 4, or may bind to the B23 polypeptide that is phosphorylated at Serine- 4. In alternative embodiments, the compound may be administered in conjunction with a toxin.

In alternative embodiments, the compound may be an antibody that specifically binds to the B23 polypeptide that is phosphorylated at Serine-4, for example, the anti-phospho-Mekl/2 antibody. In alternative embodiments, the compound the cell proliferative disease may be a cancer.

In alternative aspects, the invention provides the use of a therapeutically-effective compound that recognises a B23 polypeptide that is phosphorylated at Serine-4 for formulating

a medicament for, for example, treating a cell proliferative disease. The compound may modulate phosphorylation of a B23 polypeptide at position 4, or may bind to the B23 polypeptide that is phosphorylated at Serine-4. In alternative embodiments, the compound may be administered in conjunction with a toxin. In alternative embodiments, the compound may be an antibody that specifically binds to the B23 polypeptide that is phosphorylated at Serine-4, for example, the anti-phospho-Mekl/2 antibody. In alternative embodiments, the cell proliferative disease may be a cancer.

In alternative aspects, the invention provides a polypeptide substantially identical to a B23 polypeptide comprising an amino acid substitution at position 4. The amino acid substitution may prevent phosphorylation of the polypeptide under conditions which would normally result in phosphorylation of an unsubstituted B23 polypeptide, for example, Serine to Alanine substitution. In alternative embodiments, the substitution may mimic phosphorylation of the polypeptide, for example, a Serine to Threonine substitution, a Serine to Aspartate substitution, or a Serine to Glutamate substitution. In alternative aspects, the invention provides an isolated B23 polypeptide that is phosphorylated at Serine 4, or provides a polypeptide including a sequence selected from Accession numbers AAH50628, AAH21983, AAH21668, AAH16824, AAH16768, AAH1, 6716, AAH14349, AAH12566, AAH08495,, AAH02398, NP002511, P06748, AAP36411, AAP35657, A32915, BAB40600, AAA58386, AAA36385, AAB94739, AAH54755, NP037124, NP032748, P13084, I52858, A34168, A36089, A28939, DNCHFM, P07222, A41730, Q61937, P16039, AAA41731, AAA41730, AAA40796, AAA40795, AAA40794, or AAA39801, where the polypeptide includes a substitution at position 4.

In alternative embodiments, the substitution results in chromosome decondensation in a cell. In alternative embodiments, the invention provides a pharmaceutical composition including any of the polypeptides of the invention in combination with a physiologically acceptable carrier. In alternative embodiments, the invention provides an immunogenic fragment of any of the polypeptides of the invention, or a nucleic acid molecule encoding a polypeptide according to the invention. In alternative embodiments, the invention provides an antibody, for example, a polyclonal antibody or a monoclonal antibody, that specifically binds to a B23 polypeptide that is phosphorylated at Serine 4.

A"B23 polypeptide"is a multifunctional, highly acidic nucleolar phosphoprotein. B23 polypeptides according to the invention include, without limitation, human polypeptides identified by the following Accession numbers: AAH50628, AAH21983, AAH21668,

AAH16824, AAH16768, AAH16716, AAH14349, AAH12566, AAH08495, AAH02398, NP002511, P06748, AAP36411, AAP35657, A32915, BAB40600, AAA58386, AAA36385, AAB94739, or a fragment, analogue, or variant thereof. B23 polypeptides may also include B23 amino acid sequences from other species, for example, those identified by the following Accession numbers: AAH54755 (mouse), NP037124 (rat), NP032748 (mouse), P13084 (rat), 152858 (mouse), A34168 (rat), A36089 (rat), A28939 (rat), DNCHFM (chicken), P07222 (frog), A41730 (frog), Q61937 (mouse), P16039 (chicken), AAA41731 (rat), AAA41730 (rat), AAA40796 (rat), AAA40795 (rat), AAA40794 (rat), AAA39801 (mouse), or a fragment, analogue, or variant thereof.

"Position 4"of a B23 polypeptide refers, in general, to the serine residue that is generally found at the fourth position from the N-terminus in the B23 sequences referred to by Accession numbers herein. In some embodiments, a B23 polypeptide includes the sequence MEDSMDMDMSPLRPQNYLFGCE (SEQ ID NO: 1), or any fragment thereof, for example, MEDSMDM (SEQ ID NO: 2), that is capable of being phosphorylated by a polo-like kinase, or is capable of generating an antibody response, for example, when phosphorylated at position 4.

A B23 nucleic acid molecule encodes a B23 polypeptide, as described herein.

A"phosphorylated"B23 protein or polypeptide is post-translationally modified on any amino acid residue capable of being phosphorylated in vivo. In the context of the present invention, a phosphorylated B23 protein is generally phosphorylated on Serine 4 (Ser-4) of the amino acid sequence. An"unphosphorylated"B23 protein may be incapable of being phosphorylated on an amino acid residue capable of being phosphorylated in vivo, for example, by mutation of that residue to an amino acid that is not capable of being phosphorylated. A mutation of a serine to an alanine in a polypeptide sequence, for example, results in a protein that is not capable of being phosphorylated at that particular position in the polypeptide sequence. A B23 protein that possesses an alanine at position 4 instead of a serine is such an "unphosphorylated"B23 protein. An unphosphorylated B23 protein may also be a protein that is capable of being phosphorylated in vivo, for example on Ser-4, but is not phosphorylated due to, for example, the presence of an inhibitor, for example, a kinase inhibitor; due to an antibody that interferes with the phosphorylation site; or due to the activity of a phosphatase. A "constitutively phosphorylated"B23 protein is a protein that possesses a mutation at an amino acid residue, for example, Ser-4, that is capable of being phosphorylated in vivo, where the mutation mimics phosphorylation at that residue, and the resultant polypeptide possesses the

biological activity of a phosphorylated polypeptide. Generally, mutation of a phosphorylatable reside to a glutamic acid or aspartic acid residue results in constitutive phosphorylation.

A"polo-like kinase"or"Plk"is a serine/threonine kinase that is active during mitosis.

Plk's include kinases identified by the following Accession numbers: AAH06880 (mouse), AAH03002 (human), AAH02369 (human), NP 005021 (human), AAQ02497 (synthetic), P53350 (human), NP 741244 (worm), NP ? 41243 (worm), NP_501196 (worm), NP_491036 (worm), NP035251 (mouse), NP 058796 (rat), P34331 (worm), Q20845 (worm), Q9N2L7 (worm), A47545 (mouse), S34130 (human), BAC22692 (starfish), CAA73575 (human), CAA53536 (human), Q62673 (rat), Q07832 (mouse), AAF28314 (worm), BAB18588 (sea urchin), AAC14425 (worm), AAC14129 (worm), AAC34661 (worm), CAA74301 (trypanosome), CAE12059 (fruit fly), AAA56635 (mouse), AAA56634 (human), AAA39948 (mouse), AAA18885 (rat), and fragments, analogues, and variants thereof. In some embodiments, Plk's include Plkl, Plk2, Plk3, or Plk4 kinases), and fragments, analogues, and variants thereof.

A"biologically-active fragment"of a protein includes an amino acid sequence, found in a naturally-occurring protein, that is capable of modulating any of the functions of the protein, as described herein or known to those of ordinary skill in the art. For example, a biologically-active Plk fragment may be capable of phosphorylating a substrate, for example, a B23 polypeptide, or be capable of binding a phosphopeptide, for example, a B23 peptide phosphorylated at Ser-4. A"variant"protein includes in general a modification, for example, by deletion, addition, or substitution, of an amino acid sequence found in a naturally-occurring protein that is capable of modulating any of the functions described herein or known to those of ordinary skill in the art.

A"protein,""peptide"or"polypeptide"is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, regardless of post-translational modification (e. g. , glycosylation or phosphorylation). An "amino acid sequence","polypeptide","peptide"or"protein"of the invention may include peptides or proteins that have abnormal linkages, cross links and end caps, non-peptidyl bonds or alternative modifying groups. Such modified peptides are also within the scope of the invention. The term"modifying group"is intended to include structures that are directly attached to the peptidic structure (e. g. , by covalent coupling), as well as those that are indirectly attached to the peptidic structure (e. g. , by a stable non-covalent association or by covalent coupling to additional amino acid residues, or mimetics, analogues or derivatives

thereof, which may flank the core peptidic structure). For example, the modifying group can be coupled to the amino-terminus or carboxy-terminus of a peptidic structure, or to a peptidic or peptidomimetic region flanking the core domain. Alternatively, the modifying group can be coupled to a side chain of at least one amino acid residue of a peptidic structure, or to a peptidic or peptido-mimetic region flanking the core domain (e. g. , through the epsilon amino group of a lysyl residue (s), through the carboxyl group of an aspartic acid residue (s) or a glutamic acid residue (s), through a hydroxy group of a tyrosyl residue (s), a serine residue (s) or a threonine residue (s) or other suitable reactive group on an amino acid side chain). Modifying groups covalently coupled to the peptidic structure can be attached by means and using methods well known in the art for linking chemical structures, including, for example, amide, alkylamino, carbamate or urea bonds.

A"nucleic acid molecule"is any chain of two or more nucleotides including naturally occurring or non-naturally occurring nucleotides or nucleotide analogues. A nucleic acid molecule is"complementary"to another nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule.

A"substantially identical"sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, as, discussed herein, or by one or more non-conservative substitutions, deletion, or insertions located at positions of the sequence that do not destroy biological function as described herein. Such a sequence can be at least 60% or 75%, or more generally at least 80%, 85%, 90%, or 95%, or as much as 99% identical at the amino acid or nucleotide level to the sequence used for comparison. Sequence identity can be readily measured using publicly available sequence analysis software (e. g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.

53705, or BLAST software available from the National Library of Medicine, USA). Examples of useful software include the programs, Pile-up and PrettyBox. Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications.

An antibody"specifically binds"an antigen when it recognises and binds the antigen, for example, a B23 peptide that is phosphorylated at Ser-4, but does not substantially recognise and bind other molecules in a sample, for example, an unphosphorylated B23 peptide. Such an antibody has, for example, an affinity for the antigen which is 10,100, 1000 or 10000 times greater than the affinity of the antibody for another reference molecule in a sample.

"Mitosis"is a complex process by which a cell divides into two identical daughter cells.

Mitosis is divided into four phases: prophase, in which the nuclear membrane disappears and paired chromosomes are formed, along with polar bodies and spindle; metaphase, where the chromosomes arrange in the equatorial plan of the spindle and then separate; anaphase, where the two groups'of daughter chromosomes separate and move along the spindle; and telophase, where daughter nuclei are formed and cytokinesis or cytoplasmic division occurs, creating the two daughter cells. Much of mitosis is regulated by phosphorylation and dephosphorylation events. "Mitotic cells"in general are cells that are in the process of mitosis, i. e. , cell division, or are about to divide, i. e. , pre-mitotic. Cells may be undergoing, or about to undergo, mitosis for a variety of reasons, including due to a cell proliferative disorder, such as a cancer. Mitotic cells may be surrounded by, or present in, organs, tissue, or cells that are not undergoing or about to undergo mitosis.

A"cell proliferative disease"is a disorder or condition characterised by excessive or aberrant cell growth. When the normal function of cell survival go awry, the cause or the result can be cell degenerative or cell proliferative diseases, including cancer, viral infections, autoimmune disease/allergies, cardiovascular diseases, neurodegeneration, etc.

By a"cancer"or"neoplasm"is meant any unwanted growth of cells serving np, physiological function. In general, a cell of a neoplasm has been released from its normal cell division control, i. e. , a cell whose growth is not regulated by the ordinary biochemical and physical influences in the cellular environment. In most cases, a neoplastic cell proliferates to form a clone of cells which are either benign or malignant. Examples of cancers or neoplasms include, without limitation, transformed and immortalized cells, tumours, and carcinomas such as breast cell carcinomas and prostate carcinomas. The term cancer includes cell growths that are technically benign but which carry the risk of becoming malignant.

"Modulating"or"modulates"means changing, by either increase or decrease. The increase or decrease may be a change of any value between 10% and 90%, or of any value between 30% and 60%, or may be over 100%, when compared with a control or reference sample or compound.

A"test compound"is any naturally-occurring or artificially-derived chemical compound. Test compounds may include, without limitation, peptides, polypeptides, synthesised organic molecules, naturally occurring organic molecules, and nucleic acid molecules. A test compound can"compete"with a known compound such as a Plk protein or fragment thereof by, for example, interfering with modulation of cell proliferation, apoptosis or

cell death, phosphopeptide binding, or protein phosphorylation by a Plk or the known compound, or by interfering with any biological response induced by Plk or the known compound. Generally, a test compound will exhibit any value between 10% and 90%, or over 100%, modulation when compared to a Plk protein, peptide or peptide analogue, or other reference compound. For example, a test compound may exhibit at least 20% modulation, or at least 30% to 50% modulation, or even over 80% or over 100% modulation. A compound that is a negative modulator will in general decrease modulation relative to a known compound, while a compound that is a positive modulator will in general increase modulation relative to a known compound.

A"sample"can be any organ, tissue, cell, or cell extract isolated from a subject, such as a sample isolated from a mammal having a cell proliferative or degenerative disorder. For example, a sample can include, without limitation, tissue, cells, peripheral blood, or solid tumours, isolated from a mammal with a cancer. A sample can also include cultured cells or cell lines, for example, that are not directly isolated from a subject. A sample can also be artificially derived or synthesised.

By"contacting"is meant to submit an animal, cell, lysate, extract, molecule derived from a cell, or synthetic molecule to a test compound.

By"determining"is meant analysing the effect of a test compound on the test system.

The means for analysing may include, without limitation, antibody labelling, cell proliferation assays, immunoprecipitation, in vivo and in vitro phosphorylation assays, cell death assays, immunofluorescence assays, ELISA, ultrastructural analysis, histological analysis, or any other methods described herein or known to those skilled in the art.

By"detecting"it is intended to include determining the presence or absence of a substance or quantifying the amount of a substance. The term thus refers to the use of the materials, compositions, and methods of the present invention for qualitative and quantitative determinations. For example, detecting an increase in the level of phosphorylation of a B23 polypeptide may include determining the presence or absence of phosphorylation, or may include quantifying a change of any value between 10% and 90%, or of any value between 30% and 60%, or over 100% when compared to a control.

By"detectably labelled"is meant any means for marking and identifying the presence of a molecule, e. g. , an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably-labelling a molecule are well known in the art and include, without limitation, radioactive labelling (e. g. , with an isotope such as 32p or 35S) and

nonradioactive labelling such as, enzymatic labelling (for example, using horseradish peroxidase or alkaline phosphatase), chemiluminescent labeling, fluorescent labeling (for example, using fluorescein), bioluminescent labeling, or antibody detection of a ligand attached to the probe. Also included in this definition is a molecule that is detectably labeled by an indirect means,'for example, a molecule that is bound with a first moiety (such as biotin) that is, in turn, bound to a second moiety that may be observed or assayed (such as fluorescein- labeled streptavidin). Labels also include digoxigenin, luciferases, and aequorin.

A"therapeutically effective amount"refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction of cell proliferation or induction of cell death or apoptosis. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual being treated, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.

By"toxin"is meant an agent that has a potent cytotoxic effect. Useful toxins may be derived, for example, from bacteria and plants, and include without limitation diphtheria toxin, Staphylococcal enterotoxin-A, ribonuclease, Pseudomonas exotoxin A, Pseudomonas endotoxin, abrin, DNase I, pokeweed antiviral protein, ricin, mistletoe, gelonin, curcin, modeccin, crotin, and Shigella toxin. Since toxins generally target cells non-specifically, a toxin may be conjugated to a compound, e. g. , an antibody that is specific for phosphorylated B23 Ser-4, that is specific for a proliferating cell, for example, a cell undergoing mitosis, to create a compound-toxin fusion. Such fusions bind specifically to a proliferating cell, without substantial binding to non-proliferating cells. Linkers may be used in the compound-toxin fusion to enhance cytotoxicity and binding efficiency.

Other features and advantages of the invention will be apparent from the following description of the drawings and the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-B show phosphoprotein analysis of HeLa cells in response to nocodazole treatment. The changes in the intensity of phosphorylation signals are evident in nocodazole- treated cells (Figure 1B) as compared to serum-starved control cells (Figure 1A). The cognate targets of 31 different phosphospecific antibodies applied in the screen are listed as follows:

1. Adducin-S724 ; 2. CDK1-Y15 ; 3. CREB-S133 ; 4. ERK1/2-T202/Y204 ; 5. Gsk3a/P-S21/S9 ; 6. Gsk3cc/ß-Y279/Y216 ; 7. cJun-S73 ; 8. MEK1-S217/S221 ; 9. MEK3/6-S189/S207 ; 10. MSK1-S376 ; 11. NR1-S896 ; 12. P38a-T180/Y182 ; 13. p70 S6K-T389; 14. PKBa-T308 ; 15. PKBa-S473 ; 16. PKCa-S657 ; 17. PKCa-T638 ; 18. PKC8-T505 ; 19. PCKs-S719 ; 20. PKR-T451; 21. Rafl-S259 ; 22. RB1-S780 ; 23. RB1-S809/S811 ; 24. Rskl-T360/S364 ; 25. SAPK-T183/Y185 ; 26. Smadl-S463/S465 ; 27. Src-Y418; 28. Src-Y529; 29. STAT1-Y701 ; 30. STAT3-S727 ; 31. STAT5-S694 ; 32. pp40. Only the known phosphoproteins present in the HeLa cells are identified as numbered bands, as indicated in the list. The pp40 protein that cross-reacted with anti-phospho-Mekl/2 antibody is highlighted with a box.

Figures 2A-D are immunoblots demonstrating, that pp40 is a mitosis specific protein.

The pp40 signal was detected by immunoblotting with anti-phospho-Mekl/2 antibody (Figure 2A), and the progression of the cell cycle was monitored by immunoblotting with anti-cyclin A (Figure 2B), anti-cyclin B1 (Figure 2C), and anti-phospho-CDKl (Thr 161) (Figure 2D) antibodies.

Figure 3 demonstrates that pp40 corresponds to the nucleolar protein B23/nucleophosmin by reciprocal immunoprecipitation with anti-B23 or phospho-Mekl/2 antibody from nocodazole-treated HeLa lysate.

Figures 4A-G demonstrate that pp40 is Ser-4-phosphorylated B23. Figures 4A and 4B are immunoblots of HeLa lysates treated with nocodazole or DMSO and probed with anti- phospho-Mekl/2 (pB23) or anti-B23 (B23) antibodies, respectively. Figures 4C and 4D are immunoblots of total lysates of HeLa, A549 and MCF7 cells untreated, or treated with alkaline phosphatase, and probed with anti-phospho-Mekl/2 (pB23) or anti-B23 (B23) antibodies, respectively. Figure 4E is a sequence alignment between the antigenic peptide of anti- phospho-Mekl/2 antibody and B23 N-terminus. Figures 4F and 4G are immunoblots of anti- Flag immunoprecipitates from pcDNA-B23WT-Flag or-B23S4A-Flag-transfected HEK293, treated with nocodazole or DMSO, and probed with phospho-Mekl/2 antibody and anti-Flag antibody, respectively.

Figures 5A-B demonstrate the effects of the protein kinase inhibitors PD 98059 (30 , uM), SB 203580 (5 p1M), DRB (20 uM) or LY 294002 (10 uM) on B23 Ser-4 phosphorylation (Figure 5A) and protein levels (Figure 5B) in HeLa cell lysates. Lysate from cells treated with nocodazole only was used as the control.

Figures 6A-C demonstrate that no effect was observed for both B23S4A- (Figure 6B), and B23S4E- (Figure 6C) transfected cells on cell cycle progression, when compared with empty vector (Figure 6A), as measured by flow cytometry.

Figures 7 demonstrates the results of mimicking Ser-4 phosphorylation on chromosome decondensation. The bar graph shows the distribution of the nuclear areas in the three groups of transfected cells.

Figures 8A-B demonstrate that histone H3 Ser-10 dephosphorylation is correlated with chromosome decondensation induced by B23 Ser-4 transfection. Lysates from HEK 293 cells transfected with Flag-tagged B23S4E, B23S4A, and empty vector, were immunoblotted with anti-phospho-histone H3 (Ser-10) antibody (Figure 8A) and anti-Flag antibody (Figure 8B).

Figure 9 demonstrates that the cell cycle dependent kinase cdkl does not phosphorylate B23 directly.

Figure 10 demonstrates that both B23 and cdkl can be co-immunoprecipitated with an anti-Plkl antibody.

Figure 11 demonstrates that Plkl is the main B23-Ser-4 kinase in HeLa cell lysates.

DETAILED DESCRIPTION OF THE INVENTION The invention identifies Ser-4 as a novel phosphorylation site on the B23 polypeptide, and identifies Plk's as being responsible for phosphorylating B23 at this site. This phosphorylation, in part, leads to chromosome decondensation, which is a pre-requisite for mitotic division.

The B23 Ser-4 phosphorylation is found in tumour cells, allowing it to be used as a tumour marker. In addition, the presence of the phosphorylation in tumour cells provides a chemotherapeutic target. The identification of the Ser-4 site on B23 as a substrate for Plk's also provides a high throughput screening platform for the specific detection of Plk phosphotransferase activity by, for example, using ELISA or Western blotting techniques, which obviate the need for the use of radioactivity.

Various alternative embodiments of the invention are described below. These embodiments include, without limitation, use of B23 phosphorylated at Ser-4 to detect cell proliferation, or to assay test compounds that modulate Plk activity.

Assays Various assays, as described herein or known to one of ordinary skill in the art, may be performed to determine biological activity of a test compound. For example, modulation of phosphorylation, phosphopeptide binding, or cell proliferation may be tested as described herein or as known by one of ordinary skill in the art.

Phosphorylation assays may be conducted under conditions suitable for modulating phosphorylation. For example, Plk activity or B23 phosphorylation may be determined by kinase assays using a phosphate donor, for example, radioactive ATP, and a B23 polypeptide as a substrate. Phosphorylation assays include assays conducted under conditions that inhibit or remove phosphorylation, for example, in the presence of kinase inhibitors or phosphatases.

Plk activity may be determined, for example, using a B23 polypeptide as a substrate, and conducting ELISA, immunoblot, or immunoprecipitation assays using a phospho-specific antibody directed to the B23 Ser-4 phosphorylation. The Plk and B23 polypeptides may be synthesised using recombinant DNA technology or artificial means, or may be isolated from cells, organs, or tissues.

Various cell proliferation assays, such as those described herein or known to one of ordinary skill in the art may be used. Such assays include MTT assays; contact inhibition assays, conducted on soft agar or in an animal model, for example, to determine tumour growth; as well as multiple commercially-available cell proliferation assays.

Cell Proliferative Disorders And Reagents Thereof Cell proliferative diseases and disorders include, for example, neoplasms, such as fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioandotheliosarcoma, synoviome, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, colon carcinoma, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinome, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, lung carcinoma, small call lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astracytoma, medulloblastoma, craniopharyngloma, ependymoma, pinealoma,

hemangloblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kapos's sarcoma. Disorders of cell proliferation also include haematopoietic diseases, psoriasis, atherosclerosis, dermatological diseases, such as pemphigus vulgaris and pemphigus foleaceus, and inflammatory disorders.

Cells and tissues may be derived from subjects having any of these disorders. Cell lines used as models of proliferative diseases may include commercially available cells from, for example, the American Type Culture Collection (ATCC), Manassus, VA, USA. Such cell lines may include LnCaP cells, HeLa cells, Daudi cells, Raji cells, HEK 293 cells, etc.

Animal Models Animal models of proliferative diseases include, for example, transgenic rodents (e. g. mice, rats) bearing gain of function proto-oncogenes (e. g. Myc, Src) and/or loss of function of tumour suppressor proteins (e. g. p53, Rb) or rodents that have been exposed to radiation or chemical mutagens that induce DNA changes that facilitate neoplastic transformation. Many such animal models are commercially available, for example, from The Jackson Laboratory, ME, USA. These animal models may be used as source cells or tissue for the assays of the invention. Test compounds may also be assayed in these models.

Antibodies The compounds of the invention can be used to prepare phospho-specific antibodies to B23 Ser-4 phosphorylated polypeptides or analogues thereof, using standard techniques of preparation as, for example, described in Harlow and Lane (Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. , 1988), or known to those skilled in the art. Antibodies can be tailored to'minimise adverse host immune response by, for example, using chimeric antibodies contain an antigen binding domain from one species and the Fc portion from another species, or by using antibodies made from hybridomas of the appropriate species. In some embodiments of the invention, the antibody is not the anti-phospho-Mekl/2 (Ser 217-Ser 221) antibody. In alternative embodiments of the invention, antibodies may be raised, for example, against a peptide comprising the sequence MEDpSMDMDMSPLRPQNYLFGCE (where"pS"refers to a phosphorylated Serine residue) (SEQ ID NO: 3), or an immunogenic fragment thereof that contains the phosphorylated form of Ser-4 (or variant thereof, for example, phosphorylated Thr-4).

Polypeptides And Test Compounds In one aspect, compounds according to the invention include B23 polypeptides, for example, the B23 polypeptides described herein that are phosphorylated or unphosphorylated at the Ser-4 position, including polypeptides that are constitutively phosphorylated, or that are unphosphorylatable, as well as homologs and fragments thereof. In alternative embodiments, a compound according to the invention can be a B23 peptide or peptide analogue, for example, MEDSMDMDMSPLRPQNYLFGCE (SEQ ID NO: 1), or fragment thereof, as long as it is capable of being phosphorylated by Plk.

Compounds can be prepared by, for example, replacing, deleting, or inserting an amino acid residue at position 4 of a B23 protein, peptide or peptide analogue, as described herein, with other conservative amino acid residues, i. e. , residues having similar physical, biological, or chemical properties, and screening for the ability of the compound to act as a kinase substrate for Plk.

In alternative aspects, the test compound may be a compound, for example, a polypeptide, or variant or analog thereof, or a small molecule, that is capable of modulating phosphorylation of a B23 polypeptide at position 4.

In some embodiments of the invention, compounds of the invention include antibodies that specifically bind to a B23 polypeptide that is phosphorylated at Ser-4. In alternative embodiments, the antibody is not the anti-phospho-Mekl/2 (Ser 217-Ser 221) antibody.

It is well known in the art that some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. In one aspect of the invention, polypeptides of the present invention also extend to biologically equivalent peptides that differ from a portion of the sequence of the polypeptides of the present invention by conservative amino acid substitutions. As used herein, the term"conserved amino acid substitutions"refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.

As used herein, the term"amino acids"means those L-amino acids commonly found in naturally occurring proteins, D-amino acids and such amino acids when they have been

modified. Accordingly, amino acids of the invention may include, for example: 2-Aminoadipic acid; 3-Aminoadipic acid; beta-Alanine; beta-Aminopropionic acid; 2-Aminobutyric acid; 4- Aminobutyric acid; piperidinic acid ;, 6-Aminocaproic acid; 2-Aminoheptanoic acid; 2- Aminoisobutyric acid ; 3-Aminoisobutyric acid; 2-Aminopimelic acid; 2,4 Diaminobutyric acid; Desmosine; 2,2'-Diaminopimelic acid; 2, 3-Diaminopropionic acid; N-Ethylglycine; N- Ethylasparagine; Hydroxylysine; allo-Hydroxylysine; 3-Hydroxyproline; 4-Hydroxyproline; Isodesmosine; allo-Isoleucine ; N-Methylglycine ; sarcosine; N-Methylisoleucine; 6-N- methyllysine; N-Methylvaline; Norvaline; Norleucine; and Ornithine.

In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e. g. , within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about-1.6 such as Tyr (-1.3) or Pro (-1.6) s are assigned to amino acid residues (as detailed in United States Patent No. 4,554, 101, incorporated herein by reference): Arg (+3.0) ; Lys (+3.0) ; Asp (+3.0) ; Glu (+3.0) ; Ser (+0.3) ; Asn (+0.2) ; Gln (+0.2) ; Gly (0); Pro (-0.5) ; Thr (-0.4) ; Ala (-0.5) ; His (-0.5) ; Cys (-1.0) ; Met (-1.3) ; Val (-1.5) ; Leu (-1.8) ; Ile (-1.8) ; Tyr (-2.3) ; Phe (-2.5) ; and Trp (-3.4). <BR> <BR> <BR> <BR> <P> In alternative embodiments, conserved amino acid substitutions may be made where an<BR> amino acid residue is substituted for another having a similar hydropathic index (e. g. , within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: Ile (+4.5) ; Val (+4.2) ; Leu (+3.8) ; Phe (+2.8) ; Cys (+2.5) ; Met (+1.9) ; Ala (+1.8) ; Gly (-0.4) ; Thr (-0.7) ; Ser (-0.8) ; Trp (-0.9) ; Tyr (-1.3) ; Pro (-1.6) ; His (-3.2) ; Glu (-3.5) ; Gln (-3.5) ; Asp (- 3.5) ; Asn (-3.5) ; Lys (-3.9) ; and Arg (-4.5).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.

Conservative amino acid changes can include the substitution of an L-amino acid by the corresponding D-amino acid, by a conservative D-amino acid, or by a naturally-occurring, non- genetically encoded form of amino acid, as well as a conservative substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include beta-alanine, 3-amino- propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-amino-butyric acid,

N-methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine, t- butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, norvaline, 2- napthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine, 2- fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2, 3,4- tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diamino butyric acid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine, cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid.

In alternative embodiments, conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge. Amino acids can be generally characterized as hydrophobic or hydrophilic, depending primarily on the properties of the amino acid side chain. A hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eisenberg et al. (J. Mol. Bio.

179: 125-142,184). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, Ile, Pro, Met and Trp, and genetically encoded hydrophilic amino acids include Thr, His, Glu, Gln, Asp, Arg, Ser, and Lys. Non-genetically encoded hydrophobic amino acids, include t-butylalanine, while non-genetically encoded hydrophilic amino acids include citrulline and homocysteine.

Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amino acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents such as-OH,-SH,-CN,-F,-Cl,-Br,-I,-NO2,-NO,- NH2, -NHR,-NRR,-C (O) R, -C (O) OH, -C (O) OR, -C (O) NH2, -C (O) NHR, -C (O) NRR, etc., where R is independently (Cl-C6) alkyl, substituted (Cl-C6) alkyl, (Cl-C6) alkenyl, substituted (CI-C6) alkenyl, (CI-C6) alkynyl, substituted (CI-C6) alkynyl, (C5-C20) aryl, substituted (Cs- C20) aryl, (C6-C26) alkaryl, substituted (C6-C26) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl or substituted 6-26 membered alkheteroaryl. Genetically encoded aromatic amino acids include Phe, Tyr, and Trp, while non-genetically encoded aromatic amino acids include phenylglycine, 2-napthylalanine, beta-2- thienylalanine, 1, 2,3, 4-tetrahydro-isoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2- fluorophenylalanine3-fluorophenylalanine, and 4-fluorophenylalanine.

An apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i. e. , the side chain is not polar).

Genetically encoded apolar amino acids include Gly, Leu, Val, Ile, Ala, and Met, while non- genetically encoded apolar amino acids include cyclohexylalanine. Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala, Leu, Val, and Ile, while non-genetically encoded aliphatic amino acids include norleucine.

A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and Gln, while non-genetically encoded polar amino acids include citrulline, N-acetyl lysine, and methionine sulfoxide.

An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7.

Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His, while non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3,-diaminopropionic acid, 2,4-diaminobutyric acid, and homoarginine.

It will be appreciated by one skilled in the art that the above classifications are not absolute and that an amino acid may be classified in more than one category. In addition, amino acids can be classified based on known behaviour and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids. Amino acids can also include bifunctional moieties having amino acid- like side chains.

Conservative changes can also include the substitution of a chemically derivatised moiety for a non-derivatised residue, by for example, reaction of a functional side group of an amino acid. Thus, these substitutions can include compounds whose free amino groups have been derivatised to amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Similarly, free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or

hydrazides, and side chains can be derivatized to form O-acyl or 0-alkyl derivatives for free hydroxyl groups or N-im-benzylhistidine for the imidazole nitrogen of histidine. Peptide analogues also include amino acids that have been chemically altered, for example, by methylation, by amidation of the C-terminal amino acid by an alkylamine such as ethylamine, ethanolamine, or ethylene diamine, or acylation or methylation of an amino acid side chain (such as acylation of the epsilon amino group of lysine). Peptide analogues can also include replacement of the amide linkage in the peptide with a substituted amide (for example, groups of the formula-C (O)-NR, where R is (Cl-C6) alkyl, (C1-C6) alkenyl, (Cl-C6) alkynyl, substituted (Cl-C6) alkyl, substituted (C1-C6) alkenyl, or substituted (Cl-C6) alkynyl) or isostere of an amide linkage (for example,-CH2NH-,-CH2S,-CH2CH2-,-CH=CH- (cis and trans),- C (O) CH2-, -CH (OH) CH2-, or-CH2SO-).

The compound can be covalently linked, for example, by polymerisation or conjugation, to form homopolymers or heteropolymers. Spacers and linkers, typically composed of small neutral molecules, such as amino acids that are uncharged under physiological conditions, can be used. Linkages can be achieved in a number of ways. For example, cysteine residues can be added at the peptide termini, and multiple peptides can be covalently bonded by controlled oxidation. Alternatively, heterobifunctional agents, such as l l disulfide/amide forming agents or thioether/amide forming agents can be used. The compound can also be linked to a another compound that can modulate an apoptotic, aggregative, or proliferative response. The compound can also be constrained, for example, by having cyclic portions.

Peptides or peptide analogues can be synthesised by standard chemical techniques, for example, by automated synthesis using solution or solid phase synthesis methodology.

Automated peptide synthesisers are commercially available and use techniques well known in the art. Peptides and peptide analogues can also be prepared using recombinant DNA technology using standard methods such as those described in, for example, Sambrook, et al.

(Molecular Cloning: A Laboratory Manual. 2. sup. nd, ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. , 1989) or Ausubel et al.

(Current Protocols in Molecular Biology, John Wiley & Sons, 1994). Thus, compounds according to the invention include B23 nucleic acid molecules, for example, cDNA, RNA, genomic DNA, or antisense molecules, or fragments thereof.

In general, candidate compounds for modulating Plk activity or for detecting cell proliferation or mitosis are identified from large libraries of both natural products or synthetic

(or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the method (s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic-or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e. g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available.

Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceanographic Institute (Ft. Pierce, FL, USA), and PharmaMar, MA, USA. In addition, natural and synthetically produced libraries of, for example, pancreatic beta cell precursor polypeptides containing leader sequences, are produced, if desired, according to methods known in the art, e. g. , by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily, modified using standard chemical, physical, or biochemical methods.

When a crude extract is found to modulate cell survival, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having cell proliferation, -preventative, or-palliative activities. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value may be subsequently analyzed using a mammalian HD model, or any other animal model for a degenerative or proliferative disorder.

Candidate test compounds can be first assayed for their ability to modulate Plk kinase activity, cell proliferation or other response. The cells or cell preparations used in the assays can be obtained from cell lines or can be isolated from patients or animal models for degenerative or cell proliferative diseases or disorders, using standard techniques. The assays

can be performed using standard assays as described herein, or known to those of ordinary skill in the art. Test compounds that modulate Plk kinase catalytic activity, cell proliferation or other responses can then be used for further analysis. Test compounds identified as being modulators of Plk kinase catalytic activity, cell proliferation or other responses can be further tested in animal models of cell degeneration or proliferation, using standard techniques.

Pharmaceutical Compositions, Dosages, And Administration Compounds of the invention can be provided alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to humans. If desired, treatment with a compound according to the invention may be combined with more traditional and existing therapies for degenerative or proliferative diseases.

Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from or presymptomatic for cell proliferative diseases. Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences" (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example,

polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

For therapeutic or prophylactic compositions, the compounds are administered to an individual in an amount sufficient to stop or slow cell degeneration or cell proliferation, depending on the disorder. An"effective amount"of a compound according to the invention includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount"refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction of cell death or apoptosis, or promotion of cell proliferation, for a cell degenerative disease, or the promotion of cell death or apoptosis, or reduction of cell proliferation, for a cell proliferative disease. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.

A"prophylactically effective amount"refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as reduction of cell death or apoptosis, or promotion of cell proliferation, for a cell degenerative disease, or the promotion of cell death or apoptosis, or reduction of cell proliferation, for a cell proliferative disease.

Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. A preferred range for therapeutically or prophylactically effective amounts of a compound may be 0.1 nM-O. lM, 0.1 nM-0. 05M, 0.05 nM-151lM or 0.01 nM-101lM.

It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

In the case of vaccine formulations, an immunogenically effect amount of a compound of the invention can be provided, alone or in combination with other compounds, with an adjuvant, for example, Freund's incomplete adjuvant or aluminum hydroxide. The compound may also be linked with a carrier molecule, such as bovine serum albumin or keyhole limpet hemocyanin to enhance immunogenicity.

In general, compounds of the invention should be used without causing substantial toxicity.

Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i. e. , the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the compositions.

The examples described herein are provided as illustrations of the invention, and should not be construed as limiting the scope of the invention.

MATERIALS AND METHODS Cell Lines and Culture Conditions HeLa (human cervical carcinoma), HEK 293 (human embryonic kidney cells), MCF-7 (human breast carcinoma), and A549 (human lung carcinoma) were maintained in Dulbecco's modified Eagle medium (DMEM) or minimum essential medium (MEM), (GIBCO-BRL, Burlington, ON, Canada) supplemented with 10% fetal bovine serum (FBS, GIBCO-BRL), penicillin (100 U/ml) and streptomycin (100 ug/ml), in a humidified atmosphere containing 5% C02. The culture dishes used for HEK 293 were treated with 2% poly-L-lysine (Sigma, St.

Louis, MO, USA) for 20 min prior to each use.

Antibodies and Chemicals Anti-Phospho Mekl/2 (Ser-217/Ser-221) (CatalogNo. 9121) and anti-phospho-CDK1 (Thrl61) rabbit polyclonal antibodies were purchased from Cell Signaling Technologies (Beverly, MA, USA). Anti-human B23 goat polyclonal antibody, anti-cyclin A, anti-cyclin B1 monoclonal antibodies and HRP-conjugated secondary antibodies were from Santa Cruz

Biotechnology (Santa Cruz, CA, USA). Anti-Flag monoclonal antibody was from Sigma and anti-phospho-histone H3 (Ser-10) rabbit polyclonal antibody (Catalog No. 06-597) and active CDK1/cyclin B (Catalog No. 14-450) were purchased from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Anti-Plkl antibody (Catalog No. 610558) was from BD Transduction Labs, and anti-CDKl antibody (Catalog No. AH20122) was from Biosource International.

Protein kinase inhibitors including olomoucine, SB203580, PD98059, DRB and LY294002, and calf intestine alkaline phosphatase were from Calbiochem (San Diego, CA, USA). All other chemicals were purchased from Sigma, unless otherwise stated.

Plasmids Plasmid pGEX-4T-B23 wild-type (WT) was provided by Dr. Kenji Fukasawa (University of Cincinnati, OH, USA). The human B23 gene was released by restriction enzyme digestion from the pGEX4T vector and subcloned into pcDNA3. 1 (Invitrogen, Carlsbad, CA). pcDNA3-B23S4A-Flag and pcDNA3-B23S4E-Flag were constructed using polymerase chain reaction, by incorporating Flag tags at the C-termini and replacing Ser-4 with Ala and Glu, respectively. The resulting mutant constructs were then confirmed by DNA sequencing.

Transient Transfection HEK 293 cells were transiently transfected at 40-60% confluency with plasmids, as described herein, using Lipofectin transfection reagent from GIBCO-BRL according to the manufacturer's protocol. The transfection medium was removed and the cells were incubated in complete DMEM overnight to allow B23 expression prior to nocodazole treatment, or for 24 or 48 h before flow cytometry analysis.

KinetworA Analysis Total cell lysates were prepared as described previously (Zhang et al. , 2001). Briefly, cells were washed with ice-cold PBS, scraped in lysis buffer (20 mM Tris, 20 mM ß- glycerophosphate, 150 mM NaCI, 3 mM EDTA, 3 mM EGTA, 1 mMNa3V04, 0.5% Nonidet P-40, and 1 mM dithiothreitol) supplemented with 1 mM phenylmethanesulfonylfluoride, 2 pg/ml leupeptin, 4 Fg/ml aprotinin and 1 llg/ml pepstatin A, and sonicated for 15 sec. Cell debris was removed by centrifugation at 100,000 rpm for 30 min. at 4°C. The protein concentration was determined by the Bradford assay (Bradford, 1976). For Kinetwork

phosphoprotein screen---KPSS 1.1 or 1.2 (Pelech et al. , 2002; Kinexus Bioinformatics Corporation, Vancouver, BC, Canada), 300 or 350 zg of total protein were resolved on a 13% single lane SDS-polyacrylamide gel, and transferred to nitrocellulose membrane. Using a 20- lane multiblotter from BioRad, the membrane was then incubated with a panel of antibodies specific for phosphorylated forms of over 33 known signaling proteins of distinct molecular masses, using different mixtures of up to 3 antibodies per lane that react with a distinct subset of the proteins, followed by a mixture of relevant HRP conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were developed using ECL PlusTM reagent (Amersham Pharmacia, Piscataway, NJ, USA) and the signals were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).

Flow Cytometry B23WT-, B23S4A-and B23S4E-transfected HEK 293 cells grown in 100mm dishes were collected by trypsinization 24 or 48 hours after transfection. The cells were washed in phosphate buffered saline (PBS) once, and fixed in methanol for 30 min at-20°C. After three washes in PBS, the cells were resuspended in PBS containing 25 pg/ml RNase A and 100 llg/ml propidium iodide at 37°C for 30 min. The DNA fluorescence After three washes in PB, S, the cells were resuspended in PBS containing 25 ug/ml RNase A and 100 ug/ml propidium iodide at 37°C for 30 min. was measured using a FACSCalibur cytometer (Becton Dickinson, Mississauga, ON, Canada); data acquisition and analysis was performed with the Cell Quest software. Aliquots of flow cytometry samples were examined under a fluorescence microscope, and the sizes of the nuclei were measured using Northern Eclipse software (Enpix, ON, Canada).

Immunofluorescence microscopy B23WT-, S4A-and S4E-transfected HEK 293 cells grown on coverslips for 24 hours after transfection were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature followed by extensive washes in PBS. After being permeabilized in 0.2% Triton X-100 in PBS for 5 min, cells were incubated with anti-Flag monoclonal antibody (1: 250) in 3% BSA for 2 h at room temperature followed by Alexa488-labeled anti-mouse IgG secondary antibody (1: 1000) from Molecular Probes (Eugene, OR) for 1 h in the dark. Nuclei were counter-stained with propidium iodide following RNase digestion. Cells were observed and images were collected under a BioRad_Multiphoton confocal microscope (Bio-Rad).

Preparation of Demembranated HEK 293 Nuclei and DNA Decondensation Studies Demembranated nuclei were prepared according to the protocol of Philpott et al.

(1991), as used for mouse myeloma nuclei. Briefly, approximately 2x106 HEK 293 cells were detached from culture dishes mechanically, pelleted, washed once with PBS, and resuspended in 2 ml of low salt lysis buffer (20 mM HEPES-KOH, pH 6.8, 5 mM KC1, 5 mM MgC12, and 0.5% Nonidet P40, supplemented with protease inhibitors) on ice for 45 min (Evan and Hancock) 1985). Cells were pelleted at 1000 rpm at 4°C and rinsed twice in lysis buffer.

Demembranated nuclei were resuspended in 400 tl incubation buffer (8 mM HEPES, pH 7.5, 8 mM KC1, 2 mM MgC12, 200 mM sucrose and 50% glycerol) (Matsumoto et al. , 1999).

In a standard decondensation assay, 5 p, l of the nuclear preparation were incubated with 2 g of dialyzed GST-B23 fusion proteins or GST only at 37°C in 30 ul of incubation buffer supplemented with 3.3 mM ATP, 33 mM creative phosphate, 0.33 mg/ml phosphocreatine kinase, and 0.8 mM dithiothreitol. After 30 min incubation, 30 ul of PBS containing 7.4% formaldehyde and 100 llg/ml propidium iodide were added. Samples were then visualized under a Radiance Plus or BioRad Multiphoton confocal microscope (Bio-Rad, Hercules, CA, <BR> <BR> <BR> USA).

Phospho-B23 (Ser-4) Purification, LC-Mass Spectrometry (LC-MS), and Tandem Mass Spectrometry (MS/MS) Analyses One hundred twenty milligrams of total protein from HeLa cells, treated with 100 ng/ml nocodazole for 24 hours, were used as the starting material for purification. The purification scheme included DEAE-Sephacel, Phenyl-Sepharose, Mono Q and Mono S. At each step, the protein signal was monitored by Western blotting analysis using the anti- phospho-Mekl/2 (Ser-217/Ser-221) antibody. The Mono S fractions enriched for a 40 kDa immunoreactive protein were pooled and concentrated by ultrafiltration. The sample was then subjected to SDS-PAGE, followed by Coomassie blue staining of the gel. The 40 kDa immunoreactive protein band was excised and digested in-gel with trypsin (Promega, Madison, WI, USA). The mass spectrometry sample was prepared according to the protocol described by Kinter and Sherman (2000).

EXAMPLE 1: A Phospho-Mekl/2 (Ser-217/Ser-221) Antibody Recognizes A Mitotis-Specific 40 KDa Protein To better understand the effects of microtubule disrupting agent, nocodazole, on cell signaling pathways in mammalian cells, the KinetworkTM phospho-site screen KPSS 1.1 or 1.2, which utilizes 31 phosphorylation-site specific antibodies, was used to track changes in the phosphorylation states of 33 known cell signaling proteins in response to nocodazole treatment.

Exposing exponentially growing HeLa cells to 100 ng/ml nocodazole for 20 hours resulted in mitotic arrest induced by microtubule disruption. While a number of expected changes were observed, an approximately 40 kDa phosphoprotein (pp40) was consistently detected by an antibody that was raised against phospho-Mekl/2 (Ser-217/Ser-221) with at least 3 times higher intensity in nocodazole-treated cells, compared to untreated control cells (Figures 1A- C). The small size of pp40 precluded it from being one of the original targets of the antibody, Mekl or Mek2, which are 45 kDa in size.

To establish whether pp40 was a nocodazole-specific or a mitosis-associated protein, pp40 was monitored in serum-starved HeLa cells following serum addition. Twenty-four or forty-eight hours after serum starvation, serum was added back to the medium to induce HeLa cells to enter the cell cycle synchronously. At various time points after serum addition,, cells were harvested and total lysates prepared. A maximum level of pp40 was seen at 24 hours after serum addition, coinciding with the time when cyclin B1 was maximally expressed and CDK1 maximally phosphorylated at its Thr-161 activation site, followed by a gradual decrease (Figures 2A-D). Similarly, pp40 was also detected in HEK 293, A549 and MCF-7 cells by the same antibody, with higher abundance in mitotic cells than interphase cells, indicating that pp40 is associated with mitosis, and is not a stress response to nocodazole treatment.

EXAMPLE 2: Mass Spectrometry Identifies pp40 As B23/Nucleophosmin The partial purification of pp40 from the total lysate of nocodazole treated HeLa cells was undertaken to reveal the molecular identity of pp40.

The purification scheme consisted of sequential liquid chromatography over DEAE- Sephacel, Phenyl-Sepharose, Mono Q, and Mono S columns. The columns were developed either with linear or step NaC1 gradients. The elution profile of pp40 was monitored by Western blotting with the phospho-Mekl/2 (Ser-217/Ser-221) antibody at each step. Following Mono S, the fractions enriched for pp40 were pooled and separated by SDS-PAGE.

The band corresponding to pp40 was excised after Coomassie Blue staining and digested in-gel with trypsin. The eluted fragments were then subjected to LC-mass spectrometry. Five tryptic peptides from. LC-MS were further characterized with tandem MS (MS/MS). A Mascot database search indicated that pp40 was human B23/nucleophosmin (Table 1) (Perkins et al. , 1999). Table 1: Tryptic Peptide Analysis Mr (expt) Mr (calc) Peptide 598. 38 598. 38 VPQKK (SEQ ID NO: 4) 822. 38 822. 42 GQESFKK (SEQ ID NO: 5) 909. 43 909. 46 SKGQESFK (SEQ ID NO: 6) 971. 49 971. 50 DTPAKNAQK (SEQ ID NO: 7) 1111. 61 1111. 60 SAPGGGSKVPQK (SEQ ID NO: 8) The molecular identity of pp40 was further supported by reciprocal co- immunoprecipitation data. B23 was detectedlin phospho-Mekl/2 (Ser-217/Ser-221) antibody + immunoprecipitates by Western blotting with a B23 antibody, and pp40 was found in anti-B23 immunoprecipitates (Figure 3). Immunofluorescence microscopy with the phospho-Mekl/2 antibody revealed a signal exclusively localized within the nucleolus in interphase and translocåted into the cytoplasm during M-phase, which is consistent with B23.

EXAMPLE 3: pp40 Is B23 Phosphorvlated At The Novel Site Ser-4 Immunoblotting of HeLa lysates treated with nocodazole or DMSO with anti-phospho- Mekl/2, which recognises the Ser-217/Ser-221 phosphorylated forms of Mekl and Mek2 (Figure 4A), or anti-B34 (Figure 4B) antibodies revealed that the increase in pp40 signal was not due to a change in B23 protein level. Furthermore, treatment of lysates from nocodazole- treated HeLa, MCF-7 and A549 cells with 35 U alkaline phosphatase for 4 hours at 37°C resulted in the disappearance of pp40 (Figure 4C), whereas B23 protein levels remained relatively constant (Figure 4D), indicating the phospho-Mekl/2 antibody recognized a phospho-epitope on B23.

The human B23 full-length sequence was aligned with the antigenic phosphopeptide of the antibody using the Clustal W algorithm (Higgins et al. , 1994) to identify the phospho-

epitope on B23 recognized by the phospho-Mekl/2 antibody. The results indicated that only the flanking sequences of Ser-4 on B23 shared limited similarity with the antigenic peptide of the antibody (Figure 4E). Three consecutive residues including Ser-4 (B23)/Ser-217 (Mekl) and two adjacent residues (Asp at-1 and Met at +1 positions) were identical between the two sequences. This implicated Ser-4 as the phosphorylation site recognized by the antibody.

A non-phosphorylatable mutant of B23 (B23S4A) was constructed by replacing Ser-4 with Ala, and HEK 293 cells were transfected with either a FLAG-tagged B23S4A or FLAG- tagged B23WT construct. After 20 hours nocodazole treatment, the overexpressed Flag-tagged B23WT or B23S4A proteins were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-Flag (Figure 4G) or anti-phospho-Mekl/2 antibody (Figure 4F). As can be seen in Figure 4G, while the expression levels of B23WT and B23S4A were similar, the phospho-Mekl/2 antibody recognized two bands in B23WT- transfected cell lysates, but only the lower band was detected in B23S4A-transfected cell lysates (Figure 4F). The differential immunoreactivities of the two bands with anti-Flag and B23 antibodies indicated that the upper band corresponded to the overexpressed Flag-tagged B23 proteins, whereas the lower band corresponded to endogenous B23. The disappearance of the upper band upon replacing Ser-4 with Ala demonstrated that Ser-4 was the phosphorylation site recognized by the phospho-Mekl/2antibody. The presence of the endogenous phospho-Ser- 4 B23 in anti-Flag immunoprecipitates may be due to the formation of oligomers between overexpressed B23 and endogenous B23 proteins. Its appearance in both B23WT and B23S4A-transfected cells indicated that the Ser-4 phosphorylation is not required for all the B23 molecules involved in oligomerization, and that eliminating Ser-4 phosphorylation does not disrupt B23 oligomeric activity, even though Ser-4 is localized within the functional domain required for oligomerization (Hingorani et al. , 2000).

EXAMPLE 4: Phosphorylation Of Ser-4 Of B23 Is Indirectly Regulated By CDK's HeLa cells were treated with several protein kinase inhibitors to determine the kinase responsible for B23 Ser-4 phosphorylation. HeLa cell lysates were treated with PD 98059 (30 uM), SB 203580 (5 uM), olomoucine (100 I1M), DRB (20 I1M) or LY 294002 (10 uM), along with nocodazole (100 ng/ml) for 16 or 20 h. Lysate from cells treated with nocodazole only was used as the control.

While the inhibitors for Mekl (PD98059), p38 MAP kinase (SB203580), CK2 (DRB) and PI 3-kinase (LY294002) did not inhibit Ser-4 phosphorylation of B23, treatment with the

direct CDK inhibitor, olomoucine, resulted in complete loss of Ser-4 phosphorylation (Figure 5A), with no changes in B23 protein levels (Figure 5B), as demonstrated by Western blotting using the anti-phospho-Mekl/2antibody (pB23) and anti-B23 antibody (B23). This implicated a CDK as being responsible for B23 Ser-4 phosphorylation. Since B23 Ser-4 does not reside within a typical CDK phosphorylation site, i. e. no proline is found in + 1 position after Ser-4, the results indicated that a CDK acts on B23 indirectly by activating an upstream kinase of B23, which in turn phosphorylates, Ser-4 of B23. In keeping with this, neither purified starfish CDK1/cyclin B complex nor nocodazole-treated HeLa lysate were able to phosphorylate Ser-4 on bacterially expressed B23WT protein. Furthermore, Histone HI (detected with phospho- Histone H1 antibody), but not GST-B23 Ser4 (detected with the phospho-Mekl/2 Ser-217/Ser- 221 antibody), can be phosphorylated by active Cdkl/CyclinBl in vitro (Figure 9). This indicates that the olomoucine inhibition of B23 Ser-4 phosphorylation that was evident in nocodazole-treated HeLa cells was an indirect effect on the kinase that phosphorylates B23 Ser-4 in vivo.

EXAMPLE 5: Phosphorylation Of Ser-4 On B23 Results In Chromosome Decondensation In HEK 293 Cells The effect of Ser-4 phosphorylation on the cell cycle progression was examined, since there is a temporal correlation between Ser-4 phosphorylation of B23 with the onset of mitosis.

HEK293 cells were transfected with either pcDNA3 vector (Figure 6A), pcDNA3-B23 S4A (Figure 6B), or pcDNA3-B23S4E (Figure 6C) and harvested for flow cytometric analysis 24 hours after transfection. Flow cytometry data demonstrated that the B23S4A-and vector- transfected cells displayed very similar cell cycle distribution profiles, at least up to 24 hours after transfection (Figures 6A-B). Similarly, no effect on cell cycle progression was observed in similar experiments with another B23 mutant, B23S4E, having Ser-4 replaced by Glu to mimic Ser-4 phosphorylation (Figure 6C). The minimal cell death (sub-G 1) observed was comparable in the two types of transfected cells. The apparent lack of interference on the cell cycle by the B23 Ser-4 mutation may be due to the high abundance of endogenous B23WT, relative to the transfected B23S4A in the cells, which would mask any dominant negative effects of the B23S4A mutant.

B23 is translocated into the cytoplasm in mitotic cells with condensed chromosomes.

When in HEK 293 cells were stained for the expression of B23S4E, B23S4A, and pcDNA empty vector, and the cells were examined under a fluorescence microscope, B23S4E-

transfected cells appeared to have larger nuclei compared to B23S4A and empty vector- transfected cells, as revealed by propidium iodidine staining of the nuclei. Expression of B23S4E, but not B23S4A nor pcDNA empty vector, in HEK 293 cells for 72 h after transfection induced the expansion of nuclear areas.

Distribution of the nuclear areas in three groups of transfected cells is shown in Figure 7. Fifty-four nuclei from each group were sampled randomly for measuring nuclear area, and about 15% of nuclei sampled from the B23S4E group had nuclear areas ranging from 400 to 900 um2, which were not found in either B23S4A or vector group. Given that there was no apparent increase of the number of polyploid cells in B23S4E cells as determined by flow cytometry (Figure 6A-C), the enlargement of nuclear area in B23S4E cells could not be attributed to DNA over-replication, but could reflect chromosome decondensation.

To confirm the effect of Ser-4 phosphorylation on chromosome conformation, demembranated HEK293 nuclei were incubated with bacterially-expressed GST-B23S4A or GST-B23S4E protein. Incubation with B23S4E protein for 30 min at 37°C resulted in the appearance of dispersed nuclei, whereas no effects were seen after incubation with B23S4A or GST only. Thus, incubating demembranated HEK 293 nuclei with GST-B23S4E protein, but not GST-B23S4A or GST, resulted in decondensation of nuclei. These in vitro incubation results were consistent with the observation made in transfected cells in vivo. Furthermore, probing overexpressed B23S4E, B23S4A, and B23WT proteins with anti-Flag antibody in HEK cells revealed no difference in subcellular localization between mutant and wild type proteins in either interphase or mitotic cells.

The level of histone H3 Ser-10 phosphorylation, an event closely associated with chromosome condensation (Hendzel et al. , 1997), was monitored in B23S4E, B23S4A, and vector control transfected cells to investigate whether the expansion of nuclei in B23S4E cells was due to chromosome decondensation. As can be seen in Figure 8, the level of histone H3 phosphorylation on Ser-10 was lowest in B23S4E cells, compared to that in either B23S4A or vector control, which is consistent with the conclusion that Ser-4 phosphorylation plays a role in chromosome decondensation. Thus, mimicking Ser-4 phosphorylation by replacing the serine with a glutamic acid resulted in chromosome decondensation of HEK 293 nuclei both in vivo and in vitro, but exhibited no effect on subcellular localization of mutant proteins.

Chromosome condensation at the onset of mitosis has been postulated to be a two-step process: a transient decondensation step to allow the access of factors necessary for transcription cessation and chromosome condensation, followed by actual chromosome

condensation (Roth and Allis, 1992). The B23 Ser-4 phosphorylation regulates this transient decondensation step at the G2/M transition during the normal cell cycle, and subsequent additional phosphorylation events induce actual chromosome condensation. Thus, mitotic chromosome condensation is a coordinated effect of both B23 Ser-4 and other mitotic phosphorylation events. The chromosome decondensation observed in B23S4E transfected cells, which mimic the phosphorylation at Ser-4 of B23, is likely due to the untimely expression of the singly-Ser-4 phosphorylated B2 mimic polypeptide at interphase, without the involvement of other mitotic phosphorylation events, leading to amplification of the signal for chromosome decondensation. When these cells enter mitosis, however, the singly-Ser-4 phosphorylated B23 mimic polypeptide could be further phosphorylated at mitotic phosphorylation sites, similar to endogenous B23, leading to chromosome condensation in mitosis, and accounting for the unaffected cell cycle profile in B23S4E-transfected cells.

EXAMPLE 6 Plk Phosphorylates B23 At Ser-4 Lysates of HeLa cells were treated with nocodazole for 20 hours, which activates the polo-like kinase (Plk), and immunoprecipitated with anti-Plkl antibody. Aliquots of the total lysate and the lysate immunoprecipitated with the anti-Plkl antibody, were subjected to SDS- PAGE and immunoblotted with anti-CDK1 antibody or with the anti-phospho-Mekl/2 Ser217/Ser-221 antibody, which recognizes B23 phosphorylated at Ser-4 (Figure 10). The results indicated that, as expected, the anti-Plkl antibody was able to co-immunoprecipitate CDK1. Surprisingly, the anti-Plkl antibody was also able to co-immunoprecipitate B23 phosphorylated at Ser-4, indicating that B23 is a substrate for the kinase activity of Plk. The data are consistent with CDK1 phosphorylating and activating Plkl, which in turn phosphorylates B23 at Ser-4, and explain how an inhibitor of CDK1, e. g. , olomoucine, is able to inhibit the phosphorylation of B23 at Ser-4 in vivo, when CDK1 cannot phosphorylate this site in B23 in vitro.

In a separate set of experiments, lysates from DMSO-treated HeLa cells and those treated with 100 ng/ml nocodazole for 20 hours were in some cases pre-incubated with the anti-Plk antibody, and the Plk-depleted lysate was then assayed for its ability to phosphorylate B23 at Ser-4 in vitro, as monitored with the anti-phospho-Mekl/2 Ser217/Ser-221 antibody (Figure 11). The results indicated that nocodazole-treated cell lysates contained a kinase activity that strongly phosphorylated recombinant GST-B23 at Ser-4 (Lane 2). This activity

was successfully depleted using the anti-Plkl antibody (Lane 1), indicating that Plk was principally responsible for this phosphotransferase activity. There was no B23 Ser-4 phosphorylating activity in lysates from cells that were not in mitosis (Lane 3), which corresponds to the time when Plk is not activated. The results therefore indicate that that Plk is the main B23 Ser-4 kinase.

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OTHER EMBODIMENTS Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Accession numbers, as used herein, refer to Accession numbers from multiple databases, including GenBank, the European Molecular Biology Laboratory (EMBL), the DNA Database of Japan (DDBJ), or the Genome Sequence Data Base (GSDB), for nucleotide sequences, and including the Protein Information Resource (PIR), SWISSPROT, Protein Research Foundation (PRF), and Protein Data Bank (PDB) (sequences

from solved structures), as well as from translations from annotated coding regions from nucleotide sequences in GenBank, EMBL, DDBJ, or RefSeq, for polypeptide sequences.

Numeric ranges are inclusive of the numbers defining the range. In the specification, the word "comprising"is used as an open-ended term, substantially equivalent to the phrase"including, but not limited to", and the word"comprises"has a corresponding meaning. Citation of references herein shall not be construed as an admission that such references are prior art to the present invention. All publications are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples, drawings, and claims.