WO1999047638A2 - Mutations in mitotic check point genes and methods of diagnosis - Google Patents

Abstract

The present invention is based on the understanding of the molecular basis of MIN in human tumours as related to a similar MIN phenotype in S. cerevisiae cells harboring mutations of yeast MMR genes. Following this paradigm, the present inventors identified the basis for CIN in human tumour cells as related to mitotic checkpoint defects.

Description

MUTATIONS IN MITOTIC CHECK

POINT GENES AND METHODS OF

DIAGNOSIS

TECHNICAL FIELD

This invention relates to generally to cellular growth control and more particularly to mitotic check point genes and defects therein as related to loss of cellular growth control.

BACKGROUND

Genetic instability was one of the first characteristics postulated to underlie neoplasia. Recent experiments have demonstrated that such instability occurs in two different forms. In a small fraction of colorectal and some other cancers, defective mismatch repair (MMR) results in an increased mutation rate at the nucleotide level and consequent widespread microsatellite instability (MIN). In the majority of colorectal cancers, and probably in many other cancer types, a chromosomal instability (CIN) leading to aneuploidy is observed. The physiologic and molecular bases of this pervasive abnormality are obscure. Here we demonstrate that CIN is consistently associated with the loss of mitotic checkpoint function. Moreover, in some CIN cancers, the loss of this checkpoint was associated with the muta- tional inactivation of a human BUB 1 gene, the homologue of which is known to control mitotic checkpoints and chromosome segregation in yeast. The normal mitotic checkpoints of MIN cells became defective upon transfer of mutant hBUBl alleles from either of two CIN cancers.

The key insight leading to the discovery of the molecular basis of MIN in human tumours was the discovery of a similar MIN phenotype in S. cerevisiae cells harboring mutations of yeast MMR genes. Following this paradigm, we reasoned that the basis for CIN in human tumour cells might be mitotic checkpoint defects similar to those previously ' observed in yeast cells with chromosomal instability. Cells with such defects are expected to exit mitosis prematurely after treatment with microtubule-disrupting agents. SUMMARY

The present invention is based on the understanding of the molecular basis of MIN in human tumours as related to a similar MIN phenotype in S. cerevisiae cells harboring mutations of yeast MMR genes. Following this paradigm, the present inventors identified the basis for CIN in human tumour cells as related to mitotic checkpoint defects.

In a first embodiment, the invention provides a method of diagnosing a cell prolifera- tive disorder in a subject associated with a mitotic check point gene, comprising determining the presence of a mutant mitotic check point gene in the sample wherein an the presence of a mutant mitotic check point gene in the sample is indicative of a cell proliferative disorder. In another embodiment, the invention provides a method for identifying a compound which modulates cell proliferation, comprising contacting a sample containing a mitotic check point gene product with a compound suspected of having a mitotic check point gene product modulating activity and detecting an effect on cell proliferation.

In another embodiment, the invention provides a method of detecting a cell prolifera- tive disorder in a subject comprising, quantifying the presence of a mutant mitotic check point gene, wherein the presence of the mutant mitotic check point gene is indicative of a cell proliferative disorder.

In yet another embodiment, the invention provides a method for identifying a composition that modulates hBUB 1 activity or expression, said method comprising incubating the composition with hBUB 1 protein, or with a cell expressing hBUB 1 under conditions sufficient to allow the components to interact; and determining the effect of the substance on hBUBl activity or expression.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

Figure 1 Cell cycle analysis of MIN and CIN cells. MIN (HCT116, DLD1 , and RKO) and CIN (SW480, V400, and HT29) cells were treated with nocodazole or colcemid for 18 hours,

.?- stained with the DNA-specific dye Hoechst 33258, and analyzed by flow cytometry. and 4' refer to the DNA contents of each cell line in Gl and G2/M states, respectively. Figure 2 Mitotic indices and DNA synthesis in MIN and CIN cells. Cells were treated with nocodazole (a) or colcemid (b) for the indicated times, stained with H33258, and analyzed by fluorescence microscopy, (c) BrdU was added to the nocodazole- or colcemid-treated cultures 2.5 h prior to harvest. The bars represent the percent reduction in BrdU incorporation compared to untreated cells, assessed using an antibody to BrdU. At least 200 cells were counted for each determination and the result shown is representative of those observed in three independent experiments. Figure 3 Sequence analysis of human BUB1 genes, a, S. cerevisiae (So and human protein sequences were aligned using Macaw Version 2.0.3. CD1, NLS, and CD2 represent sequence blocks that were highly related among the genes (see text). Identical amino acids are shaded^, b, Sequence of hBUB 1 in cell lines V400 and V429 and in normal cells and the primary tumours from the patients from whom the corresponding cell lines were derived. Arrows indicate the G to A transition at the canonical splice donor site following codon 40 in V400 and the C to A transversion at codon 492 in V429. Figure 4 hBUBl expression system. Following transfection of expression vectors encoding

mutant hBUB 1 from V400 (Bubl *V400) or a control vector without hBUB l, into HCT1 16

cells, FACS was used to isolate transfected cells on the basis of GFP expression. Cells were

analyzed with fluorescence microscopy to ensure that the sorted cells expressed GFP (left

panels), and to analyze mitotic indices following H33258 staining (middle panels) and cell

cycle distributions (right panels). Arrows point to cells in mitosis. The cell cycle distribu_¬

tions before (red profiles) and after (black profiles) nocodazole treatment are shown on the right. T and 4' are defined in Fig. 1.

Figure 5 Effects of hBUB l expression. Mitotic indices (a) or BrdU incorporation (b) in

nocodazole-treated, hBUB 1 -transfected cells were compared with that of control-transfected cells. Transfected cells were isolated through cell sorting on the basis of GFP fluorescence_. The bars and brackets represent the means and standard deviations, respectively, determined

from at least two independent assessments of 100 cells each in a single experiment; similar

results were obtained in two independent experiments. The hBUB 1 expression vectors (wt or

mutant from V400 ["*400"] or V429 ["*429"]), and the cell lines transfected, are indicated.

DETAILED DESCRIPTION

The present invention is based on the understanding of the molecular basis of MIN in human tumours as related to a similar MIN phenotype in S. cerevisiae cells harboring mutations of yeast MMR genes. Following this paradigm, the present inventors identified the basis for CIN in human tumour cells as related to mitotic checkpoint defects.

DIAGNOSTIC TECHNIQUES

The invention provides a method for detecting a cell proliferative disorder associated with a mitotic check point gene (e.g., BUB1, BUBRl) in a tissue of a subject, comprising contacting a target cellular component suspected of expressing a mitotic check point gene (e.g., BUB1, BUBRl) or having a mitotic check point gene associated disorder, with a reagent which binds to the component. The target cell component can be a nucleic acid, such as DNA or RNA, or a protein, or a polypeptide. When the component is a nucleic acid, the reagent is a polynucle- otide probe or PCR primer. When the cell component is a polypeptide, the reagent is an antibody probe. The probes can be detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody or probe, or will be able to ascertain such, using routine experi- mentation.

For purposes of the invention, an antibody or nucleic acid probe specific for a mitotic check point gene (e.g., BUB1, BUBRl) may be used to detect the presence of a wild-type or mutant mitotic check point gene polypeptide (using an antibody) or poiynucleotide (using a nucleic acid probe) in a biological fluid or tissue. Oligonucleotide primers based on any coding sequence region in the mitotic check point gene or polynucleotide sequence are useful for amplifying polynucleotide encoding the mitotic check point gene, for example by PCR. Any specimen containing a detectable amount of polynucleotide or antigen can be used. One such sample source may be colorectal tissue or a colorectal specimen. Alternatively, a biological fluid such as blood may be used which may contain cell indicative of a mitotic check point gene-associated cell proliferative disorder. The subject may be any organism, such as a mammal, but is preferably a human.

A number of techniques known to those skilled in the art may be used to detect a polynucleotide encoding a mitotic check point gene. For example, RNAse protection assays may be used if RNA is the polynucleotide obtained from the sample. In this procedure, a labeled antisense RNA probe is hybridized to the complementary polynucleotide in the sample. The remaining unhybridized single-stranded probe is degraded by ribonuclease treatment. The hybridized, double stranded probe is protected from RNAse digestion. After an appropriate time, the products of the digestion reaction are collected and analyzed on a gel (see for example Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, section 4.7.1 (1987)). As used herein, "RNA probe" refers to a polynucleotide capable of hybridizing to RNA in a sample of interest. Those skilled in the art will be able to identify and modify the RNAse protection assay specific to the polynucleotide to be measured, for example, probe specificity may be altered, hybridization temperatures, quantity of nucleic acid as well as other variables known to those skilled in the art. Additionally, a number of commercial kits are available, for example, RiboQuantTM Multi-Probe RNAse Protection Assay System (Pharmingen, Inc., San Diego, CA).

Additionally, detection of a polynucleotide encoding a mitotic check point gene may be performed by standard methods such as size fractionating the nucleic acid. Methods of size fractionating the DNA and RNA are well known to those of skill in the art. such as by gel electrophoresis, including polyacrylamide gel electrophoresis (PAGE). For example, the gel may be a denaturing 7 M or 8 M urea-polyacrylamide-formamide gel. Size fractionatine the nucleic acid may also be accomplished by chromatographic methods known to those of skill in the art.

The detection of a polynucleotide may optionally be performed by using radioactively labeled probes. Any radioactive label may be employed which provides an adequate signal. Other labels include ligands, which can serve as a specific binding pair member for a labeled ligand, and the like. The labeled preparations are used to probe for a polynucleotide by the Southern or Northern hybridization techniques, for example. Nucleotides obtained from samples are transferred to filters that bind polynucleotides. After exposure to the labeled polynucleotide probe^, which will hybridize to nucleotide fragments containing target nucle_ic acid sequences, the binding of the radioactive probe to target nucleic acid fragments is identified by autoradiography (see Genetic Engineering, 1, ed. Robert Williamson. Academic Press (1981), pp. 72-81). The particular hybridization technique is not essential to the invention. Hybridization techniques are well known or easily ascertained by one of ordinary skill in the art. For example, RNA is separated on a gel transferred to nitrocellulose and probed with complementary DNA to the sequence of interest. The complementary probe may be labeled radioactively, chemically etc. Hybridization of the probe is indicative of the presence of the polynucleotide of interest. The relative intensity of a reporter attached to the probe, for example a radionucleotide, can be indicative of the amount of mitotic check po_int gene or polynucleotide present in the sample. As improvements are made in hybridization techniques, they can readily be applied in the method of the invention.

The polynucleotides encoding a mitotic check point gene product or polypeptide may be amplifⁱed before detecting. The term "amplified" refers to the process of making multiple copies of the nucleic acid from a single polynucleotide molecule. The amplification of polynucleotides can be carried out in vitro by biochemical processes known to those of skill in the art. The amplification agent may be any compound or system that will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I_, Taq polymerase. Klenow fragment of E^. coli DNA polymerase I. T4 DNA polymerase. other available DNA poiv- merases, polymerase muteins, reverse transcriptase, ligase, and other enzymes, including heat-stable enzymes (i.e., those enzymes that perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation). Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products that are complementary to each mutant nucleotide strand. Generally, the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths. There may be amplification agents, however, that initiate synthesis at the 5' end and proceed in the other direction, using the same process as described above. In any event, the method of the invention is not to be limited to the embodiments of amplification described herein.

One method of in vitro amplification which can be used according to this invention is the polymerase chain reaction (PCR) described in U.S. Patent Nos. 4,683.202 and 4,683,195. The term "polymerase chain reaction" refers to a method for amplifying a DNA base sequence using a heat-stable DNA polymerase and two oligonucleotide primers, one complementary to the (+)-strand at one end of the sequence to be amplified and the other complementary to the (-)-strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. The polymerase chain reaction is used to detect the presence of polynucleotides encoding cytokines in the sample. Many polymerase chain methods are known to those of skill in the art and may be used in the method of the invention. For example, DNA can be subjected to 30 to 35 cycles of amplification in a thermocycler as follows: 95 °C for 30 sec, 52° to 60 °C for 1 min, and 72 °C for 1 min, with a final extension step of 72 °C for 5 min. For another example, DNA can be subjected to 35 polymerase chain reaction cycles in a thermocycler at a denaturing temperature of 95°C for 30 sec, followed by varying annealing temperatures ranging from 54-58°C for 1 min, an extension step at 70°C for 1 min and a final extension step at 70°C. The primers for use in amplifying the polynucleotides of the invention may be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof so long as the primers are capable of hybridizing to the polynucleotides of interest. One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition. The primer must prime the synthesis of extension products in the presence of the inducing agent for amplification.

Primers used according to the method of the invention are complementary to each strand of nucleotide sequence to be amplified. The term "complementary" means that the primers must hybridize with their respective strands under conditions which allow the agent for polymerization to function. In other words, the primers that are complementary to the flanking sequences hybridize with the flanking sequences and permit amplification of the nucleotide sequence. Preferably, the 3' terminus of the primer that is extended has perfectly base paired complementarity with the complementary flanking strand.

Those of ordinary skill in the art will know of various amplification methodologies which can also be utilized to increase the copy number of target nucleic acid. The polynucleotides detected in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific nucleic acid sequence such as another polymerase chain reaction, oligomer restriction (Saiki et al, Bio/Technology 3: 1008-1012 (1985)), allele- specific oligonucleotide (ASO) probe analysis (Conner et al, Proc. Natl. Acad. Sci. USA 80: 278 (1983), oligonucleotide ligation assays (OLAs) (Landegren et al, Science 241: 1077

(1988)), RNAse Protection Assay and the like. Molecular techniques for DNA analysis have been reviewed (Landegren et al, Science, 242: 229-237 (1988)). Following DNA amplification, the reaction product may be detected by Southern blot analysis, without using radioactive probes. In such a process, for example, a small sample of DNA containing the polynuc- leotides obtained from the tissue or subject are amplified, and analyzed via a Southern blotting technique. The use of non-radioactive probes or labels is facilitated by the high level of the amplified signal. In one embodiment of the invention, one nucleoside triphosphate is radioactively labeled, thereby allowing direct visualization of the amplification product by autoradiography. In another embodiment, amplification primers are fluorescently labeled and run through an electrophoresis system. Visualization of amplified products is by laser detection followed by computer assisted graphic display.

Simple visualization of a gel containing the separated products may be utilized to determine the presence or expression of a mitotic check point polynucleotide and mutant polynucleo- tides. For example, staining of a gel to visualize separated polynucleotides may be used, a number of stains are well known to those skilled in the art. However, other methods known to those skilled in the art may also be used, for example scanning densitometry, computer aided scanning and quantisation as well as others.

Another technique which may be used to detect a mitotic check point gene product involves the use of antibodies. Such antibodies may consist of an antibody coupled to a low molecular weight hapten. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl, pyridoxal, and fluorescein, which can react with specific antihapten antibodies.

The method for detecting a cell expressing a mutant mitotic check point gene product or a cell proliferative disorder associated with a mitotic check point gene product, described above, can be utilized for detection of colorectal cancer, breast cancer or other malignancies in a subject, including those in a state of clinical remission. Additionally, the method for detecting a mitotic check point polypeptide in cells is useful for detecting a cell proliferative disorder by measuring the level of mutant mitotic check point polynucleotides or polypep- tides in cells or in a suspect tissue, wherein the presence of such mutants is indicative of a cell proliferative disorder. Using the method of the invention, mitotic check point gene expression can be identified in a cell and the appropriate course of treatment can be employed (e.g., antisense gene therapy or drug therapy). The expression pattern of a mitotic check point gene may vary with the stage of malignancy of a cell proliferative disorder or neoplasm, therefore, a sample such as breast tissue or colorectal tissue can be screened with a panel of mitotic check point gene-specific reagents (i.e., nucleic acid probes or antibodies to mitotic check point polynucleotide or polypeptide) to detect mitotic check point gene expression and diagnose the stage of malignancy of the cell.

Monoclonal antibodies used in the method of the invention are suited for use. for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the monoclonal antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

The term "immunometric assay" or "sandwich immunoassay", includes simultaneous sandwich, forward sandwich and reverse sandwich immunoassays. These terms are well understood by those skilled in the art. Those of skill will also appreciate that antibodies according to the present invention will be useful in other variations and forms of assays which are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.

Monoclonal antibodies can be bound to many different carriers and used to detect the presence of a mitotic check point gene product. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation.

For purposes of the invention, a mitotic check point gene product may be detected by the monoclonal antibodies when present in biological fluids and tissues. Any sample containing a detectable amount of a mitotic check point gene product can be used. A sample can be a liquid such as blood, serum and the like, or a solid or semi-solid such as tissues, feces. and the like, or, alternatively, a solid tissue such as those commonly used in histological diagnosis.

In performing the assays it may be desirable to include certain "Mockers" in the incubation medium (usually added with the labeled soluble antibody). The "Mockers" are added to assure that non-specific proteins, proteases, or anti-heterophilic immunoglobulins to anti- mitotic check point gene product immunoglobulins present in the experimental sample do not cross-link or destroy the antibodies on the solid phase support, or the radiolabeled indicator antibody, to yield false positive or false negative results. The selection of "Mockers" therefore may add substantially to the specificity of the assays described in the present invention.

It has been found that a number of nonrelevant (i.e., nonspecific) antibodies of the same class or subclass (isotype) as those used in the assays (e.g., IgGl, IgG2a, IgM, etc.) can be used as "Mockers". The concentration of the "Mockers" (normally 1-100 μg/μl) may be important, in order to maintain the proper sensitivity yet inhibit any unwanted interference by mutually occurring cross reactive proteins in the specimen.

IN Vivo IAGNOSTTΓS

-I I- Monoclonal antibodies may be used for the in vivo detection of antigen, the detectably labeled monoclonal antibody is given in a dose which is diagnostically effective. The term "diagnostically effective" means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having a mitotic check point gene product antigen for which the monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which is administered should be sufficient such that the binding to those cells having a mitotic check point gene product is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio. Typically, the monoclonal antibody is readioactively labelled, however, other labels are within the scope of the invention, for example, paramagnetic isotopes.

As a general rule, the dosage of detectably labeled monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. The dosage of monoclonal antibody can vary from about 0.001 mg/m² to about 500 mg/m², preferably 0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m² to about 10 mg/m². Such dosages may vary, for example, depending on whether multiple injections are given, tumor burden, and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting a given radioisotope. The radioisotope chosen must have a type of decay which is detectable for a given type of instrument. Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized. Ideally, a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140- 250 keV range, which may be readily detected by conventional gamma cameras. For in vivo diagnosis, radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to immunoglobulins are the bifunctional chelating agents such as diethylenetriaminepentacetic acid (DTP A) and ethylene- diaminetetraacetic acid (EDTA) and similar molecules. Typical examples of metallic ions which can be bound to the monoclonal antibodies of the invention are '"In. ⁹⁷Ru. ^6;Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and ²⁰¹T1.

A monoclonal antibody useful in the method of the invention can also be labeled with a para- magnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ^l62Dy, ⁵²Cr, and ⁵⁶Fe.

Monoclonal antibodies used in the method of the invention can be used to monitor the course of amelioration of a mitotic check point gene-associated cell proliferative disorder. Thus, by measuring the increase or decrease in the number of cells expressing a mutant mitotic check point gene or changes in mitotic check point gene expression present in various body fluids. such as blood or serum, it would be possible to determine whether a particular therapeutic regiment aimed at ameliorating the disorder is effective.

Detection of the mitotic check point allele

In addition, the invention may be used to identify or treat individuals who are "at risk" of developing a mitotic check pint gene-associated disorder or transmitting an allele carrying a mutant mitotic check point gene to their offspring. These individuals may be identified by a method of the invention for detecting the presence or absence of a mutant mitotic check point gene or by any other diagnostic means, and/or may be treated by a method of the invention "Homozygous" is defined as two of the same alleles for a given gene. According to the present invention, the mitotic check point gene (wt or mt), like most eukaryotic genes, at a frequency of two copies per genome. If both copies are genetically the same, the individual is homozygous, i.e. either wt/wt or mt/mt.

"Heterozygous" is defined as two different alleles being present in the genome for a given gene. According to the present invention, if one copy each of wt and mt mitotic check point gene are present in the genome, the individual is heterozygous.

Using the techniques discussed above, it is possible to identify samples and subjects which may have or be at risk of having a cell proliferative disorder or neoplasm. For example, where the sample contains nucleic acid sequences detection of a mutation in a mitotic check point gene or polynucleotide sequence is indicative of a cell proliferative disorder. In one embodiment, a method of diagnosing a cell proliferative disorder in a subject associated with a mitotic check point gene is provided. The method includes determining the presence of a mutant mitotic check point gene in a sample wherein the presence of a mutant mitotic check point gene in the sample is indicative of a cell proliferative disorder. For example, such mutations include a mutation in the mitotic check point gene "BUB 1 " wherein a deletion removing codons 76-141 of BUB 1 results in a lack of mitotic arrest. In this respect, hybrid- ization techniques and amplification techniques which hybridize or amplify the polynucleotide sequence corresponding to codons 76-141 would be capable of identifying the presence of the deletion and thus a cell proiferative disorder. In another embodiment, a missense mutation at codon 492 of BUB 1 also results in lack of mitotic arrest. Accordingly, hybridization techniques and amplification techniques can be used to focus on the polynucleotide sequence coding for codon 492, thus identifying the presence of the mutation and indicating a cell proliferative disorder.

Such techniques are also applicable to the mitotic check point gene "BUBRl ". Hybridization and amplification techniques can be utilized to identify, for example, a mutation of C to T at codon 40 of BUBRl . The mutation results in a substitution of methionine for threonine at codon 40. Furthermore a deletion of T at codon 1023 of BUBRl has been indicative of a cell proliferative disorder.

Where the sample contains a polypeptide encoded by a mitotic check point gene, the detection of mutation in the gene product (e.g., BUB 1 or BUBRl gene produce) or polypeptide can be used as indicators of a cell proliferative disorder. For example, and not by way of limitation, antibodies which recognize antigenic determinants corresponding to BUB 1 codons 76-141, or codon 492 can be used as indicators of a mutant mitotic check point polypeptide. Similarly, and not by way of limitation, antibodies which recognize antigenic determinants corresponding to BUBRl codons 40 and 1023 can be used as indicators of a mutant mitotic check pint polypeptide.

SCREENING FOR MITOTIC CHECK POINT POLYPEPTIDE BINDING COMPOUNDS Also included is a method of identifying compounds that bind mitotic check point polypeptide or fragments thereof. The method for identifying a compound which binds to mitotic check point polypeptide comprises incubating a test compound and mitotic check point polypeptide under conditions sufficient to allow the compound and mitotic check point polypeptide to form a complex. Conditions will vary according to a number of factors well recognized in the art, including temperature, concentration, hydrophobicity and hydrophilicity of the molecules, for example hydrophobic molecules such as long chain fatty acids may require buffers including dimethylsulfoxide (DMSO). Compounds can thus be any number of molecules including polypeptides, peptidomimetics, carbohydrates, fatty acids, and/or steroids. The molecules may be labeled. For example the mitotic check point polypeptide or the test compound may be chemically labeled with a fluorescence compound, a radioactive element or a metal chelating agent. The method also involves separating a complex of mitotic check point polypeptide and the binding compound from unbound mitotic check point polypeptide and measuring the binding or effect of binding of the compound to mitotic check point polypeptide. Separation of the compounds and mitotic check point polypeptide can be accomplished by any number of means including chromatography, gel electrophoresis and other well known to those skilled in the art.

The invention provides methods for decreasing cell proliferation. Cells can be treated with hBUB 1 or hBUBRl to decrease cellular proliferation. In particular, for example, a cell proliferative disorder associated with an insufficient amount of expression or activity, a therapeutic approach which directly effects the actions or activity of hBUB 1 or hBUBRl is possible. For example, nucleic acids that encode hBUBl or hBUBRl polypeptides or active fragments thereof can be introduced into cells to decrease cellular prolifera_tion. Thus, the invention provides methods for treating disorders associated with undesirable cell proliferation and abnormal cell proliferation.

The invention further provides methods for increasing cell proliferation. Antagonists of hBUB 1 or hBUBRl, such as antisense nucleic acids, ribozymes or dominant negative mutant alleles, such as the hBUB 1 V400 and V429 mutant alleles disclosed herein can be used to inhibit hBUBl or hBUBRl activity. Similarly, active fragments of such dominant negative mutant alleles can be useful for inhibiting hBUBl or hBUBRl activity. Thus, by inhibiting hBUBl or hBUBRl activity, cells can be induced to undergo mitosis and subsequent proliferation. Antisense nucleic acids (e.g. RNA or DNA molecules) can bind specifically to a targeted gene's RNA message, inhibiting the expression of that gene's protein product. Alternatively, hBUBl mutant alleles V400 or V429 can inhbit the function of wild type hBUBl thereby promoting mitososi and cell proliferation. Thus, the invention provides methods for treating disorders associated with insufficient cell proliferation or undesirable cell degeneration.

Disorders that can be treated include those associated with undesirable cell proliferation or those that are associated with insufficient cell proliferation. For example_, a sub_ject that exhibits undesirable cell proliferation, including fibrotic disorders, hyperplasias and neopla- sia can be treated by adminstering the compositions of the invention. For example. hBUB 1 or hBUBRl polypeptides. or active fragments thereof, or polynucleotides that encode them_. can be administered to a subject afflicted with a fibrotic disorder, hyperplasia or a neoplasia. In contrast, a subject that exhibits disorders associated with insufficient cell proliferation can be treated by adminstering a dominant negative hBUBl or hBUBRl mutant allele, such as the hBUB 1 V400 and V429 mutant alleles.

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded. Antisense oligo- mers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target producing cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal.Biochem., 122:289, 1988). Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al, Antisense Res. andDev., 1(3):227. 1991; Helene, C, Anticancer Drug Design, 6[6}:569, 1991). Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J.Amer.Med. Assn., 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated. There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff. Nature, 234:585, 1988) and "hammerhead "-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while "hammerhead "-type ribozymes recognize base sequences 1 1-18 bases in length. The longer the recognition sequence, the greater the likeli- hood that the sequence will occur exclusively in the target mRNA species. Consequently. hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences. These and other uses of antisense methods to inhibit the in vivo translation of genes are well known in the art (e.g., De Mesmaeker, et al, 1995. Backbone modifications in oligonucleotides and peptide nucleic acid systems. Curr. Opin. Struct. Biol 5:343-355; Gewirtz, A.M., et al, 1996b. Facilitating delivery of antisense oligodeoxynucleotides: Helping antisense deliver on its promise; Proc. Nat I. Acad. Sci. U.S.A. 93:3161-3163; Stein, CA. A discussion of G-tetrads 1996. Exploiting the potential of antisense: beyond phosphorothioate oligodeoxynucleotides. Chem. and Biol. 3:319-323).

The term "neoplasia" as used herein, encompasses any carcinoma in tissues of a subject. Such neoplasias include colon, breast, head and neck carcinomas, for example, carcinoma of the mouth, esophagus, throat, larynx, thyroid gland, tongue, lips, salivary glands, nose, paranasal sinuses, nasopharynx, superior nasal vault and sinus tumors, esthesioneuroblastoma, squamous call cancer, malignant melanoma, sinonasal undifferenti- ated carcinoma (SNUC) or blood neoplasia. Also included are carcinoma's of the regional lymph nodes including cervical lymph nodes, prelaryngeal lymph nodes, pulmonary juxtaesophageal lymph nodes and submandibular lymph nodes.

The invention provides methods for identifying compositions that modulate hBUB 1 or hBUBRl expression or activity. The methods comprise incubating the composition with hBUBl or hBUBRl protein, or with a cell expressing hBUBl or hBUBRl, under conditions that allow the components to interact, and determining the effect of the composition on hBUBl or hBUBRl activity or expression.

Compositions that modulate hBUBl or hBUBRl activity may include peptides, peptidomimetics, polypeptides, chemical compounds and biologic agents. hBUB 1 and hBUBRl activity can be assayed using methodology as described in the present Examples. For example, a test compound can be applied to a cell under conditions that allow contact between the test compound and hBUBl or hBUBRl . A compound that inhibits mitosis may increase or promote hBUBl or hBUBRl expression or ac^f"'ity. Alternatively, a test compound that increases mitosis (i.e. proliferation) may inhibit hBUBl or hBUBRl expression or activity. Contacting, as used herein, includes in solution and in solid phase, or in a cell, in vitro or in vivo. The test compound may optionally be a combinatorial library for screening a plurality of compounds. Compounds identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et al, Bio/Technology, 3: 1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, et al. Proc. Natl. Acad Sci. USA, 80:278, 1983), oligonucleotide ligation assays (OLAs) (Landegren, et al, Science, 241:1077, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren, et al, Science, 242:229-237, 1988).

Where the cell proliferative disorder is the result of under expression or reduced hBUBl or hBUBRl activity, agonists that increase hBUBl or hBUBRl activity may be used to provide a therapeutic for such cell proliferative disorders. For example, hBUBl or hBUBRl mutations that effect the activity of the protein or the expression of the genes may be overcome by administering wild-type hBUBl or hBUBRl, or active fragments thereof to a cell or to a subject. For example, polynucleotides encoding hBUB 1 or hBUBRl , or active fragments thereof, wherein the polypeptide comprises hBUBl or hBUBRl, or active f agments thereof may be delivered to a subject or a cell requiring such therapy.

Delivery of the compositions of the invention that inhibit or increase cell proliferation including antisense, triplex agents, ribozymes. competitive inhibitors, proteins, polypeptides, polynucleotides encoding hBUB 1 or hBUBRl , or active fragments thereof, and the various compositions that modulate the activity or expression of hBUBl or hBUBRl can be achieved using a variety of methods available in the art. For example, polynucleotides (e.g. gene therapy, antisense etc.) can be delivered into a cell using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus. herpes virus, vaccinia, or. preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a polynucleotide sequence of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include but are not limited to Ψ2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium Another targeted delivery system for polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 um can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al, Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al, Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination of phospholipids, particularly Wgh-phase-transition-temperature phospholipids. usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidyletha- nolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. The targeting of iiposomes has been classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, or- gan-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. In general, the compounds bound to the surface of the targeted delivery system will be ligands and receptors which will allow the targeted delivery system to find and "home in" on the desired cells. A ligand may be any compound of interest which will bind to another compound, such as a receptor.

The compositions and therapeutic agents useful in the methods of the invention can be administered to the subject parenterally by injection or by gradual perfusion over time. Administration may be intravenously, intiaperitoneally, intramuscularly, subcutaneously. intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium cMoride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents and inert gases and the like.

.99- Antibodies to hBUBl or hBUBRl polypeptides or fragments thereof could be valuable as diagnostic and therapeutic tools to aid in the detection of diseases in which hBUBl or hBUBRl is a pathological factor. Therapeutically, antibodies or fragments of the antibody molecule could also be used to neutralize the biological activity of hBUBl or hBUBRl in diseases where the activity or expression of hBUBl or hBUBRl is involved in the cell degeneration or in disorders associated with insufficient cell proliferation. Such antibodies can recognize an epitope of hBUBl or hBUBRl, or fragments thereof, for antibody recognition and neutralization of hBUBl or hBUBRl activity. As used in this invention, the term "epitope" refers to an antigenic determinant on an antigen, such as a hBUBl or hBUBRl peptide, to which the paratope of an antibody, such as an hBUBl or hBUBRl -specific antibody, binds. Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Preparation of an antibody requires a substantially purified moiety that can provide an antigenic determinant. The term "substantially pure" as used herein refers to hBUB 1 or hBUBRl, or variants thereof, which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. Substantially purified or "isolated" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. One skilled in the art can isolate hBUBl or hBUBRl or fragments thereof using standard techniques for protein purification, such as immunoprecipitation with antibodiues that bind hBUBl or hBUBRl. The purity of the hBUBl or hBUBRl peptides and fragments thereof can also be determined by amino-terminal amino acid sequence analysis. hBUBl or hBUBRl peptides include functional fragments of the peptide, as long as the activity of hBUBl or hBUBRl remains. Such activity includes, for example, the ability to arrest cell mitosis, and the ability of a dmininant negative hBUBl or hBUBRl allele to promote or increase cell mitosis and cell proliferation. Smaller peptides containing the biological activity of hBUB 1 or hBUBRl are included in the invention. As used inherein, the term "antibody" includes, in addition to conventional antibodies, such protein fragments that have the ability to recognize specifically and bind the hBUB 1 or hBUBRl polypeptides or variants thereof. Regions of the gene that differ at the protein level are well defined. A protein can be raised by expression of the wild-type gene or of the variants, or, preferably, fractions therefore. For example, the nucleic acid sequence can be cloned into expression vectors. According to this embodiment, the sequence of interest can first be obtained by employing PCR, as described above, or from a synthetic gene construction with overlapping and ligated synthetic oligonucleotides. Another alternative would involve synthesis of a short peptide. All those methodologies are well known to one skilled in the art. See, for example. Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Volumes 1 and 2 (1987), with supplements, and Maniatis et al . MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratory.

The genetic sequence encoding hBUB 1 or hBUBRl can be expressed in any known, commercially available systems. Vectors for subcloning the sequence of interest, and subsequent expression into bacterial, yeast, baculovirus, insect, or tissue culture are well known to one skilled in the art. The subcloning process could, according to one embodiment, produce a fused protein with a short N-or C-terminal extension to facilitate subsequent purifications on columns or by use of antibodies or as a localization sequence. Alternatively. the protein of interest is purified by standard protein purification protocols. See for example PROTEIN PURIFICATION -PRINCIPLES AND PRACTICE, Springer Varlag publ., New- York; and PROTEIN BIOTECHNOLOGY, Humana Press, Totowa, NJ.

The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al, Production of Polyclonal Antiser a, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al. Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992), which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See, for example. Kohler & Milstein, Nature 256:495 (1975); Coligan et al, sections 2.5.1-2.6.7; and Harlow et al, ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor Pub. 1988), which are hereby incorporated by reference. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes^, fusing the B lymphocytes with myeloma cells to produce hybridomas. cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purifⁱed from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan et al, sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al, Purification of Immuno globulin G (IgG). in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (Humana Press 1992_). Methods of in vitro and in vivo multiplication of monoclonal antibodies are well-known to those skilled in the art. Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a cont_inuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g_., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.

The antibodies are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize antibodies of the invention are competitive and non- competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric₎ ass^ Detection of the antigens using the antibodies of the invention can be done utilizing i- mmunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation. The antibodies can be bound to many different carriers and used to detect the presence of an antigen comprising the peptide of the invention. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to ascertain such, using routine experimentation.

Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl, puridoxal, and fiuorescein, which can react with specific antihapten antibodies.

The antibodies may be immunoreactive with hBUBl or hBUBRl peptide or functional fragments thereof. Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al, Nature, 256:495, 1975). The term antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab')₂, Fv and SCA fragments which are capable of binding an epitopic determinant on hBUB 1 or hBUBRl . Alternatively, a therapeutically or diagnostically useful anti-hBUB 1 or hBUBRl antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Natl Acad. Sci. USA 86: 3833 (1989), which is hereby incorporate din its entirety by reference. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al. , Nature 321 : 522 (1986); Riechmann et al. , Nature 332: 323 (1988); Verhoeyen et al, Science 2J9: 1534 (1988); Carter et al, Proc. Nat 'l Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit. Rev. Biotech. XI: 437 (1992); and Singer et al, J. Immunol 150: 2844 (1993), which are hereby incorporated by reference.

Antibodies may be derived from human antibody fragments isolated from a combina- torial immunoglobulin library. See. for example, Barbas et al , METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 1 19 (1991); Winter et al, Ann Rev. Immunol. 1: 433 (1994), which are hereby incorporated by reference. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA). In addition, antibodies may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be sued to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7: 13 (1994); Lonberg et al, Nature 368:856 (1994); and Taylor et al., Int. Immunol 6: 579 (1994), which are hereby incorporated by reference. As is mentioned above, antigens that can be used in producing hBUB 1 or hBUBRl - specific antibodies include hBUB l or hBUBRl peptides or fragments thereof. The polypeptide or peptide used to immunize an animal can be obtained by standard recombinant. chemical synthetic, or purification methods. As is well known in the art. in order to increase immunogenicity, an antigen can be conjugated to a carrier protein. Commonly used carriers include keyhole limpet hemocyanin (KLH). thyroglobulin. bovine serum albumin CBS A ). and tetanus toxoid. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit). In addition to such carriers, well known adjuvants can be administered with the antigen to facilitate induction of a strong immune response.

The invention includes polypeptides having an amino acids sequence substantially the same as the amino acid sequences of hBUB 1 or hBUBRl set forth in Figure 3 A and

GenBank Accession AF047471, or functional fragments thereof, or amino acid sequences that are substantially identical to these sequences By "substantially the same" or "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 80%, preferably 85%, more preferably 90%, and most preferably 95% homology to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 1 10 nucleotides. By "substantially identical" is also meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein assayed, (e.g., as described herein). Homology is often measured using sequence analysis software (e.g. , Sequence

Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Minor modifications of the hBUB 1 or hBUBRl primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the hBUB 1 or hBUBRl polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the biological activity of hBUB 1 or hBUBRl still exists (e.g. the modified polypeptides can arrest mitosis or. in the case of a dominant negative mutant allel, can promote or increase cell mitosis. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule which would have broader utility. For example, one can remove amino or carboxy terminal amino acids which are not required for hBUB 1 or hBUBRl biological activity.

Nucleic acid sequences of the invention can be obtained by several methods. For example, DNA can be isolated using hybridization techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences, 2) polymerase chain reaction (PCR) on genomic DNA or cDN A using primers capable of annealing to the DNA sequence of interest, and 3) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features.

Preferably the hBUBl or hBUBRl polynucleotides of the invention are derived from a mammalian organism, and most preferably from human. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oiigopeptide stretches of amino acid sequence must be known. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization of the target DNA to that single probe in the mixture which is its complete complement (Wallace, et al, Nucl Acid Res., 2:879, 1981; Maniatis, et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. 1989).

The development of specific DNA sequences encoding hBUBl or hBUBRl can also be obtained by: 1) isolation of double-stranded DNA sequences from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA. Of the three above-noted methods for developing specific DNA sequences for use in recombinant procedures, the isolation of genomic DNA isolates is the least common. This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides due to the presence of introns.

The synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known. When the entire sequence of amino acid residues of the desired polypeptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the synthesis of cDNA sequences. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid-or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the polypeptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al, Nucl. Acid Res., 11:2325. 1983).

A cDNA expression library, such as lambda gtl 1, can be screened indirectly for hBUBl or hBUBRl peptides having at least one epitope. using antibodies specific for hBUBl or hBUBRl . Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of hBUB l or hBUBRl cDNA.

DNA sequences encoding hBUBl or hBUBRl can be expressed in vitro by DNA transfer into a suitable host cell. "Host cells" are cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. In the present invention, the hBUBl or hBUBRl polynucleotide sequences may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the hBUBl or hBUBRl genetic sequences. Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al. Gene, 5J5:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem.. l63:351X. 1988) and baculovirus-derived vectors for expression in insect cells. The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).

Polynucleotide sequences encoding hBUBl or hBUBRl can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.

Transformation of a host cell with recombinant DNA may be carried out by conven- tional techniques as are well known to those skilled in the art. Where the host is prokarvotic. such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCL method using procedures well known in the art. Alternatively, MgCL or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired. When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, e- lectroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Εukaryotic cells can also be cotransformed with DNA sequences encoding the hBUB 1 or hBUBRl of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein, (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.

In yet another embodiment, the invention provides a method of treating a subject having or at risk of having a cell proliferative disorder associated with expression of hBUBl or hBUBRl comprising administering to a cancerous cell of the subject, a reagent that augments hBUBl or hBUBRl activity or expression. Administration of the reagent can be in vivo or ex vivo.

A number of embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES Cell culture and cell cycle analysis. The derivation and growth conditions of the colorectal

8 23 24 cancer cell lines used in this study have been described ' ' . The ten cell lines analyzed for mitotic checkpoint status were analyzed in detail for karyotype and MMR, as described The four MIN lines (HCT116, DLD 1 , RKO, and S W48) were MMR-deficient and diploid while the six CIN lines were MMR-proficient and aneuploid, with substantial chromosome number variability and modal chromosome numbers of 71, 119, 86, 82, 96, and 40 in lines HT29, SW480, V400. V429, Caco2, and SW837, respectively. Primary fibroblasts from normal human skin were obtained from Clonetics and used at third passage, when their doubling time was ~ 26 h, similar to that of the cancer cell lines. Nocodazole or colcemid was added to the media to final concentrations of 0.2 ug/ml or lug/ml, respectively. Cells were harvested at six hour time intervals thereafter, then fixed with glutaraldehyde and stained with Hoechst 33258 (H33258). Viable cells were defined as those without fragmented nuclei or other signs of apoptosis. Flow cytometry (> 10,000 cells per sample) was used to evaluate the cell cycle profile and fluorescence microscopy was used to assess the mitotic index (% of viable cells arrested in mitosis). In some experiments, BrdU (10 uM) was added to the cells 2.5 h before harvest and BrdU incorporation was evaluated using a monoclonal antibody to BrdU (Boehringer-Mannheim) and a rhodamine-coupled goat anti- mouse secondary antibody (Pierce).

Cloning ihBubl and hBUBRl. Full length protein and nucleotide sequences of S. cerevisiae and murine BUB1 genes were used to search the National Center for Biotechnology Information expressed sequence tag database (dbEST). Multiple overlapping EST clones were identified for the C-terminal ends of the proteins. To derive the remainder of the sequences, RACE was performed using colonic adeno carcinoma RACE-ready cDNA

(Clontech). RT-PCR amplification and sequencing from multiple cDNA samples derived from human tissues provided corrections to the contig assembled from the databases and EST clones. Genomic clones of each gene were identified by PCR-based screening of a human BAC library (Release III, Research Genetics). FISH was performed on human prometaphase spreads as described²⁵. Mutational Analysis. CIN status of evaluated lines was documented by the presence of aneuploidy or the absence of MIN ' . Two lines (LoVo and VI 394) were found to exhibit both CIN and MIN, a phenomenon noted previously . Total RNA was purified and used for

27 DNA synthesis with random hexamers and Superscript II (Life Technologies) as described The following primers were used to amplify hBUBl in three overlapping segments: 5'- CATGGACACCCCGGAAAATGTC-3 and 5'-GCATCTTTGCTGGCCACTGC-3' for codons 8 to 398; 5'-GTGGAGACATCCCATGAGGATC-3' and 5'- GGATCTTCTGCAATGGCAGCG-3' for codons 31 1 to 704; and 5'- AGCCCAAAACAGGCCTTGTCG-3' and 5'-CAGTGTGATTTTTAAGGACTGTC-3' for codons 668 to 1085 The following primers were used to amplify hBUBRl in three overlapping segments: 5'-TGTAGCTCCGAGGGCAGG-3^' and 5'- TTGAGAGCACCTCCTACACG-3' for codons 1 to 243; 5'-

GAGTCTTCTGTACCACAACG-3' and 5'-AAAACGGAGCTGCTTCTGGC-3' for codons 225 to 611; 5'-TCACAGGCTTCAGAAATGTAAC-3' and 5'- ATACATGGTGATCATAAGAGAAC-3' for codons 601 to 1050. The RT-PCR products were purified on an agarose gel and sequenced with a series of internal primers using Thermosequenase (Amersham). The intron-exon structures of selected regions of hBUBl and hBUBRl were determined through sequence analysis of PCR products generated from genomic DNA. Genomic PCR was performed using primers derived from sequences surrounding the mutations described in the text. DNA from paraffin blocks of the primary

28 tumours from which these lines were derived was purified as described previously . The sequences of all primers used for mutational analysis are available from the authors upon request. hBUBl expression vectors. pBI-GFP was constructed by insertion of the blunt-ended Hindlll/Notl fragment of phGFP-S65T (Clontech) into the EcøRV site of pBI (Clontech). pBI-GFP-Bubl wt was constructed by cloning RT-PCR products representing the wt hBUBl sequence into the Notl and Sail sites of pBI-GFP. pBI-GFP-Bubl *400 and pBI-GFP- Bub 1 *429 were constructed by insertion of RT-PCR products from tumour V400 and V429, respectively, into pBI-GFP. Vector DΝA was used to cotransfect HCT116 or DLD1 cells together with DΝA from a plasmid driving the expression of the tTA transcriptional activator (Clontech) and Lipofectamine Plus (Life Technologies). Twelve hours after transfection, cells were treated with 0.2 ug/ml nocodazole. BrdU was added to the media 15.5 hours later and the cells harvested 2.5 hours following BrdU addition. Successfully transfected cells were isolated by sorting on the basis of GFP fluorescence. Sorted cells were analyzed by flow cytometry and fluorescence microscopy as described above. BrdU incorporation was compared to that in transfected cells not treated with nocodazole.

EXAMPLE 2

Cells with such defects are expected to exit mitosis prematurely after treatment with

12.13 microtubule-disrupting agents ' . To test this hypothesis in human colorectal cancer cells, four MIN lines (HCTl 16, DLDl, RKO, and SW48) and six CIN lines (SW480, HT29, V400,

V429, Caco2, and SW837) were treated with nocodazole. As expected, all lines achieved nearly complete cell cycle blocks shortly after nocodazole treatment, with DNA contents of

4C (with 2C representing the Gl state; representative examples in Fig. 1). Morphologic analysis of the 4C-blocked cells, however, revealed a striking difference. All MIN cell lines had a normal checkpoint response, resulting in an accumulation of cells with condensed chromosomes characteristic of a sustained mitotic block. In the CIN lines, there was an abnormal response, with many fewer mitotic cells and no clear peak observed at any time point (Fig. 2a). The response of MIN cells was characteristic of those with intact mitotic

12 checkpoints , and a similar response was observed in normal human fibroblasts (Fig. 2a).

Consistent differences in mitotic indices were also observed in CIN cells vs. MIN cells after treatment with colcemid (Fig. 1 and Fig. 2b), an agent which blocks microtubules through a different mechanism than nocodazole. This defect was further established by the higher fraction of CIN cells which synthesized DNA during nocodazole or colcemid treatment (Fig. 2c). There was little overlap between the responses observed in MIN vs. CIN cells in these assays. For example, the decrease in BrdU incorporation ranged from 61 % to 92% in MIN cells and 0% to 34% in CIN cells. Similarly, the maximal mitotic indices achieved following 12-18 hours of colcemid treatment ranged from 72% to 89% in the four MIN lines, while that in the six CIN lines was 20% to 31% (p<0.01 by two-tailed Student's t- Test).

To elucidate the genetic mechanisms underlying the checkpoint defect in CIN cells, we first chose to evaluate the human homologue of S. cerevisiae BUB1. BUB1 is the prototype member of a family of genes, some of which bind to the kinetochore and all of

14-17 which are required for a normal mitotic delay in response to spindle disruption . The human homologue o BUBl was cloned and the complete coding sequence determined using a combination of DNA database searches and reverse-transcriptase polymerase chain reaction

(RT-PCR) methods. Comparison with the S. cerevisiae BUB1 gene showed that they contained two highly conserved domains (CD1 and CD2, Fig. 3a). CD1 (codons 21 to 152) directs kinetochore localization and binding to Bub3 while CD2 (codons 732 to 1043)

15,18 contains its kinase domain . A genomic clone containing the hBUBl gene was isolated and used to map its location to chromosome 2ql2-14 through fluorescence in situ hybridization (FISH).

We then selected 19 colorectal cancer cell lines of CIN phenotype to search for mutations in hBUBl through RT-PCR-mediated amplification and direct sequencing of its entire coding region. RT-PCR analysis of V400 (one of the lines whose phenotype is exhibited in Figs. 1 and 2) revealed a small amplification product (-800 bp) in addition to one of normal size (-1000 bp). Sequencing of the shorter RT-PCR product showed that it was the result of an internal 197 bp deletion predicted to remove codon 76 through codon 141 and create a frame-shift immediately thereafter. Sequencing of the relevant region of genomic DNA identified a G to A transition at the canonical splice donor site comprising the first intronic nucleotide following codon 140 (Fig. 3b). Sequence analysis of cDNA of another line, V429, revealed a missense mutation at codon 492, resulting in the substitution of tyrosine for a conserved serine (Fig. 3b). In both V400 and V429, the mutations were heterozygous and the second allele was wild-type (wt). Analysis of DNA derived from archived tissues of the patients from whom these cell lines were derived revealed that the mutations were somatic, present in their primary tumours but not in their normal tissues (Fig. 3b). To determine whether the mutant alleles in V400 and V429 could be functionally distinguished from null or wt alleles, we expressed them in MIN cells and evaluated the cells' behavior following microtubule disruption. Expression vectors encoding both a hBUBl cDNA (either wt or one of the two mutants) and a green fluorescent protein (GFP) gene were constructed and transfected into MIN cell lines. Cells were treated with nocodazole and— sorting productively-transfected cells were isolated by cell sorting on the basis of GFP fluorescence (Fig. 4). Over 90% of transfected cells were found to accumulate a DNA content of 4C following nocodazole treatment, regardless of which hBUBl sequences were present in the vector (examples in Fig. 4). However, expression of either of the mutant hBUBl genes substantially altered the mitotic indices of transfected cells after nocodazole treatment, while the wt hBUBl gene had little effect compared to a control vector devoid of hBUBl sequences (Fig. 5a). These results demonstrated that both mutants could confer a dominant negative effect in two different MIN cell lines, at least when overexpressed in comparison to endogenous levels. To determine whether a dominant negative effect was observed when mutant and wt genes were expressed at equivalent levels, cotransfections were performed. Both hBUBl mutant expression vectors resulted in altered checkpoint status when transfected with an equal amount of the wt hBUBl expression vector; the mitotic index was decreased by 45% to 69% compared to transfection with the wt hBUBl expression vector alone.

If the hBUBl mutants caused a premature exit from mitosis in the presence of microtubule disruption, one would expect that such cells would re-enter S phase, just as did the CIN cells depicted in Fig. 2c. To address this point, BrdU was added to the culture media of nocodazole-treated, transfected HCTl 16 cells 2.5 hours prior to harvest. As shown in Fig. 5b, there was significantly less reduction in BrdU incorporation after transfection with either of the two mutant hBUBl expression constructs than with the wt hBUBl expression construct or the control vector. A similar re-entry into S phase was observed in DLD 1 cells after expression of mutant hBUBl genes (Fig. 5b).

As many genes control the mitotic checkpoint in yeast, we searched for additional genes that might play a role in this process in humans. Another BUB1 homologue, named hBUBRl. was identified and its sequence determined through a strategy similar to that described above for hBUBl (Fig. 3a). The hBUBRl gene was of comparable size but was less homologous to the murine BUB1 gene than hBUBl (29% vs. 81% identical residues in the conserved domains, respectively). Sequence comparisons also revealed a third domain between CD1 and CD2 that contained a putative nuclear localization signal (NLS) that was present in both human homologues and in murine BUB1 but not in the yeast gene (Fig. 3a). A BAC clone containing hBUBRl was isolated and used in FISH experiments to show that the gene was located on human chromosome 15ql4-21. The hBUBRl gene, like hBUBl, was found to be expressed in all 19 CIN cancers analyzed. Using RT-PCR products as templates for sequencing, we noted several variants of hBUBRl that were likely to be polymorphisms based on their frequency. We additionally found two mutations, neither one of which was found among 40 normal alleles. The first was a germ-line C to T transition at codon 40, resulting in a substitution of methionine for threonine, in V531. The second mutation, found in VI 394, was a somatic deletion of T at codon 1023. Though this latter mutation was

19 predicted to remove part of the kinase domain within CD2 , further studies will be required to determine whether either of these hBUBRl mutations functionally alter the gene product.

The results described above indicate that all tested CIN cell lines were defective in a well-defined mitotic checkpoint and had a phenotype similar to that observed in yeast cells with genetic alterations of mitotic checkpoint genes such as BUB1. While these results, plus the mutational analyses reported above, do not prove that aneuploidy can be due to defects in mitotic checkpoints, the data are consistent with this possibility. The hypothesis is supported by the fact that the expression of either of two naturally-occurring hBUBl mutants converted the normal checkpoint status of MIN cells to the defective type characteristic of CIN cells.

Analogously, it has been shown that expression of in vz^'tro-generated variants of murine

18 BUB1 allowed cells to exit mitosis prematurely . Such dominant negative effects are intriguing in light of the previous demonstration that the CIN phenotype is dominant upon fusion of MIN to CIN cells .

It will be important to determine whether defects in other mitotic checkpoint genes contribute to mitotic checkpoint defects and aneuploidy in other human cancers. It is interesting in this regard that the expression of one such gene (hMAD2) was relatively low in

20 breast cancer cells that exhibited an abnormal mitotic checkpoint . Finally, the purposeful activation of cell cycle checkpoints by exogenous agents might be used to exploit the differences between normal and tumour cells described here and thus improve the selectivity

. , . 16,21,22 of chemotherapy

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Claims

We claim:

1. A method of diagnosing a cell proliferative disorder in a subject associated with a mitotic check point gene, comprising: determining the presence of a mutant mitotic check point gene in the sample wherein an the presence of a mutant mitotic check point gene in the sample is indicative of a cell proliferative disorder.

2. The method of claim 1 , wherein the cell proliferative disorder is a neoplasm.

3. The method of claim 2, wherein the neoplasm is breast or colorectal neoplasm.

4. The method of claim 1. wherein the subject is a mammal.

5. The method of claim 4, wherein the mammal is a human.

6. The method of claim 1 , wherein the mitotic check point gene is BUB 1

7. The method of claim 6, wherein the mutation is a deletion.

8. The method of claim 7, wherein the deletion removes codons 76-141 of

9. The method of claim 6, wherein the mutation is a missense mutation.

10. The method of claim 9, wherein the missense mutation is at codon 492 of BUB 1

11. The method of claim 1 , wherein the mitotic check pint gene is BUBRl

12. The method of claim 11, wherein the mutation is a C to T transition at codon 40.

13. The method of claim 11. wherein the mutation results in a substitution of methio- nine for threonine at codon 40.

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14. The method of claim 11 , wherein the mutation is a deletion.

15. The method of claim 14, wherein the deletion is a deletion of T at codon 1023 of BUBRl.

16. The method of claim 1 , wherein the presence of the mutant mitotic check point gene is determined by measuring DNA, RNA, or a polypeptide.

17. A method for identifying a compound which modulates cell proliferation, comprising contacting a sample containing a mitotic check point gene product with a compound suspected of having a mitotic check point gene product modulating activity and detecting an effect on cell proliferation.

18. The method of claim 17, wherein the compound is a peptide.

19. The method of claim 18, wherein the compound is a peptidomimetic.

20. The method of claim 18, wherein the sample comprising the mitotic check point gene product is expressed in a cell.

21. The method of claim 18, where in the compound is an antibody.

22. A method of detecting a cell proliferative disorder in a subject comprising, quantifying the presence of a mutant mitotic check point gene, wherein the presence of the mutant mitotic check point gene is indicative of a cell proliferative disorder.

23. The method of claim 22, wherein the subject is a mammal.

24. The method of claim 23 , wherein the subject is a human.

25. The method of claim 22. wherein the quantity is detected by using a probe.

26. The method of claim 25, wherein the probe is a nucleic acid.

27. The method of claim 26, wherein the nucleic acid is RNA or DNA.

28. The method of claim 25, wherein the probe is an antibody.

29. The method of claim 28, wherein the antibody is labeled.

30. The method of claim 22, wherein the cell proliferative disorder is a neoplasm.

31. The method of claim 30, wherein the neoplasm is selected from the group consisting of head. neck. lung, esophageal. stomach, small bowel, colon, bladder, kidney, breast and cervical neoplasms.

32. The method of claim 22, wherein the mitotic check point gene is BUB1 or BUBRl .

33. A method for detecting a mutant mitotic check point polypeptide in a sample, comprising: a) contacting the sample with an antibody that binds to the polypeptide; and b) detecting binding of the antibody to the polypeptide, wherein binding is indicative of the presence of the mutant polypeptide in the sample.

34. The method of claim 33, wherein the polypeptide comprises BUB 1 having a deletion at codons 76-141.

35. The method of claim 33, wherein the mutation is a missense mutation at codon 492 of BUB 1.

36. The method of claim 33, wherien the polypeptide comprises having a C to T transition at codon 40.

37. The method of claim 36, wherien the polypeptide has a subsitution of methionine for threonine at codon 40.

38. The method fo claim 33, wherein the mutation is a deletion of T at codon 1023 of BUBRl .

39. The method of claim 33, wherein the sample is a tissue.

40. The method of claim 33, wherein the sample is a biological fluid.

41. A kit useful for the detection of a mutant mitotic check point polypeptide comprising carrier means containing therein one or more containers wherein a first container contains an antibody which binds to a mutant mitotic check point polypeptide.

42. The kit of claim !, wherein the antibody is detectably labeled.

43. The kit of claim ! ! , wherein the label is selected from the group consisting of radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, and an enzyme.

44. A kit useful for the detection of a mutant mitotic check point polynucleotide comprising carrier means containing therein one or more containers wherein a first container contains a nucleic acid probe that hybridizes to the mutant mitotic check point polynucleotide.

45. The kit of claim 44, wherein the probe is detectably labeled.

46. The kit of claim 45, wherein the label is selected from the group consisting of radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, and an enzyme.

47. A method for increasing the proliferation of a cell, comprising contacting the cell with a composition that inhibits hBUBl expression or protein activity.

48. The method of claim 47, wherein said hBUBl expression or protein activity is inhibited with an inhibitor.

49. The method according to claim 48, wherein said inhibitor is selected from the group consisting of an hBUBl antibody, an hBUBl antisense nucleotide sequence, a ribozyme, a V400 mutant allele and a V429 mutant allele.

50. A method for identifying a composition that modulates hBUBl activity or expression, said method comprising: incubating the composition with hBUBl protein, or with a cell expressing hBUBl under conditions sufficient to allow the components to interact; and determining the effect of the substance on hBUB 1 activity or expression.

51. A method for increasing the proliferation of a cell in a subject, said method comprising administering to the subject in need thereof, a therapeutic amount of a mutant hBUBl or active fragment thereof effective to promote cell mitosis.

52. A method for treating a cell degenerative disorder in a subject, said method comprising administering to the subject in need thereof, a therapeutic amount of a mutant hBUBl or active fragment thereof effective to promote cell mitosis.

53. The method of claims 51 or 52, wherein said mutant hBUBl is selected from the group consisting of the V400 and V429 mutant alleles.

54. A method for treating a cell proliferative disorder in a subject, said method comprising administering to the subject in need thereof, a therapeutic amount of hBUBl or active fragment therof effective to induce mitotic arrest.

55. The method of claim 54, wherein said hBUBl or active fragment therof comprises a polypeptide.

56. The method of claim 55, wherein said polypetide comprises the polypetide sequence shown in GenBank Accession AF047471.

57. The method of claim 55, wherein said active fragment comprises a polypeptide selected from the group consisting of hBUBl CD1 and hBUBl CD2 as shown in Figure 3A.

58. The method of claim 54, wherein said hBUBl or active fragment therof comprises a nucleic acid.

59. The method of claim 58, wherein said nucleic acid comprises the nucleic acid sequence shown in GenBank Accession AF047471.

60. The method of claim 54, wherein said cell proliferative disorder is selected from the group consisting of a fibrotic disorder, benign hyperplasia, and neoplasia.

61. The method of claim 60, wherein said neoplasia is a cancer of the colon.

62. The method of claim 60, wherein said neoplasia is a cancer of the breast.

63. A method for increasing the proliferation of a cell, comprising contacting the cell with a composition that inhibits hBUBRl expression or protein activity.

64. The method of claim 63, wherein said hBUBRl expression or protein activity is inhibited with an inhibitor.

65. The method according to claim 64, wherein said inhibitor is selected from the group consisting of an hBUBRl antibody, an hBUBRl antisense nucleotide sequence and a ribozyme.

66. A method for identifying a composition that modulates hBUBRl activity or expression, said method comprising: incubating the composition with hBUBRl protein, or with a cell expressing hBUBRl under conditions sufficient to allow the components to interact; and determining the effect of the substance on hBUBRl activity or expression.

67. A method for increasing the proliferation of a cell in a subject, said method comprising administering to the subject in need thereof, a therapeutic amount of a mutant hBUBRl or active fragment thereof effective to promote cell mitosis.

68. A method for treating a cell degenerative disorder in a subject, said method comprising administering to the subject in need thereof, a therapeutic amount of a mutant hBUBRl or active fragment thereof effective to promote cell mitosis.

69. A method for treating a cell proliferative disorder in a subject, said method comprising administering to the subject in need thereof, a therapeutic amount of hBUBRl or active fragment therof effective to induce mitotic arrest.

70. The method of claim 69, wherein said hBUBRl or active fragment therof comprises a polypeptide.

71. The method of claim 70, wherein said polypetide or active fragment therof comprises the polypetide sequence shown in Figure 3A as hBUBRl .

72. The method of claim 71, wherein said active fragment comprises a polypeptide selected from the group consisting of hBUBRl CDl and hBUBRl CD2 as shown in Figure 3A.

73. The method of claim 69, wherein said hBUBl or active fragment therof comprises a nucleic acid.

74. The method of claim 69, wherein said cell proliferative disorder is selected from the group consisting of a fibrotic disorder, benign hyperplasia, and neoplasia.

75. The method of claim 74, wherein said neoplasia is a cancer of the colon.

76. The method of claim 74. wherein said neoplasia is a cancer of the breast.

77. The method of any of claims 51, 52, 54, 67, 68, or 69, wherein said subject is an animal.

78. The method of of claim 77, wherein said animal is a human.

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SUBSTITUTE SHEEKHULE 26)