Mammalian checkpoint proteins polypeptides and encoding sequences thereof

Abstract

The present invention relates to the isolation of gene sequences encoding mammalian cell cycle checkpoints, as well as the expression of the encoded proteins using recombinant DNA technology. The expressed proteins are used to generate specific antibodies and to inhibit the growth of cells. The human checkpoint gene sequences are used as a probe for a portion of the chromosome associated with tumors and other malignancies, as well as growth and/or development deficiencies.

Description

This application is a Division Application of U.S. patent application Ser. No. 08/924,183 filed on Sep. 5, 1997, now U.S. Pat. No. 6,218,109.

FIELD OF THE INVENTION

The present invention relates to mammalian proteins and gene sequences involved in cellular responses to DNA damage. In particular, the present invention provides checkpoint genes and proteins.

BACKGROUND OF THE INVENTION

The proper development of a multicellular organism is a complex process that requires precise spatial and temporal control of cell proliferation. Cell proliferation in the embryo is controlled via an intricate network of extracellular and intracellular signaling pathways that process growth regulatory signals. This signaling network is superimposed upon the basic cell cycle regulatory machinery that controls particular cell cycle transitions.

Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions, and ensure that critical events such as DNA replication and chromosome segregation are completed with high fidelity. For example, proliferating eukaryotic cells arrest their progression through the cell cycle in response to DNA damage. This arrest is critical to the survival of the organism, as failure to repair damaged DNA can result in the formation and transfer of mutations, damaged chromosomes, cancer, or other detrimental effects. The mechanism responsible for monitoring the integrity of the organism's DNA and preventing the progression through the cell cycle when DNA damage is detected is referred to as the “DNA damage checkpoint.”

In response to DNA damage, cells activate a checkpoint pathway that arrests the cell cycle, in order to provide time for repair, and induces the transcription of genes that facilitate the needed repair. In yeast, this checkpoint pathway consists of several protein kinases including phosphoinositide (Pt)-kinase homologs hATM (human), scMec1 (Saccharomyces cerevisiae), spRad3 (Schizosaccharomyces pombe), and protein kinases scDun1 (Saccharomyces cerevisiae), scRad53 (Saccharomyces cerevisiae), and spChk1 (Schizosaccharomyces pombe) (See e.g., S. Elledge, Science 1664 [1996])

Indeed, the ability to coordinate cell cycle transitions in response to genotoxic and other stressors is critical to the maintenance of genetic stability and prevention of uncontrolled cellular growth. Loss of a checkpoint gene leads to genetic instability and the inability of the cells to deal with genomic insults such as those suffered as a result of the daily exposure to ultraviolet radiation. The loss of negative growth control and improper monitoring of the fidelity of DNA replication are common features of tumor cells. When checkpoints are eliminated (e.g., by mutation or other means), cell death, infidelity in chromosome transmission, and/or increased susceptibility to deleterious environmental factors (e.g., DNA-damaging agents) result. A variety of abnormal cells arising due to infidelity during mitoses have been detected in humans, including aneuploidy, gene amplification, and multipolar mitoses (See, L. H. Hartwell and T. A. Weinert, Science 245:629 [1989]).

Accordingly, elucidation of checkpoint function, as well as the disruption of checkpoint function, will further the understanding of the process of cellular transformation (i.e., the conversion of normal cells to a state of unregulated growth), as well as cell differentiation and organismal development.

SUMMARY OF THE INVENTION

The present invention provides mammalian proteins and gene sequences involved in cellular responses to DNA damage. In particular, the present invention provides Chk1 genes and proteins.

In one embodiment, the present invention provides the nucleotide sequence set forth in SEQ ID NO:1. In alternative embodiments, the present invention provides SEQ ID NO:1, wherein it further comprises 5′ and 3′ flanking regions. In yet another embodiment, the sequence further comprises intervening regions. In a further embodiment, the present invention also provides a polynucleotide sequence which is complementary to SEQ ID NO:1 or variants thereof. In a preferred embodiment, the present invention provides a vector comprising the nucleotide sequence of claim 1. The present invention also provides host cell(s) containing the vector of claim 4.

The present invention also provides a purified Chk1 protein encoded by the nucleotide sequence of claim 1, as well as a purified protein comprising the amino acid sequence set forth in SEQ ID NO:3. In addition, the present invention provides fusion proteins comprising a least a portion of the human Chk1 protein, as well as non-Chk1 protein sequences. It is not intended that the fusion proteins of the present invention be limited to any particular portion of the Chk1 portion or any particular non-Chk1 protein sequences. In preferred embodiments, the fusion the Chk1 protein portion of the fusion protein comprises at least a portion of SEQ ID NO:3. In an alternative embodiment, the non-Chk1 protein sequence comprises an affinity tag. In particularly preferred embodiment, the affinity tag comprises a histidine tract.

In yet another embodiment, the present invention provides the sequence set forth in SEQ ID NO:2. In an alternative embodiment, the nucleotide sequence further comprises 5′ and 3′flanking regions. In another alternative embodiment, the nucleotide sequence further comprises intervening regions. In yet another embodiment, the present invention provides a polynucleotide sequence that is complementary to SEQ ID NO:2 or variants thereof.

The present invention also provides a vector comprising the nucleotide sequence set forth in SEQ ID NO:2. In one preferred embodiment, the present invention provides a host cell containing the vector comprising this nucleotide sequence.

The present invention further provides a purified Chk1 protein encoded by the nucleotide sequence of SEQ ID NO:2. In yet another embodiment, the present invention provides a purified protein comprising the amino acid sequence set forth in SEQ ID NO:4.

The present invention also provides fusions proteins comprising at least a portion of the murine Chk1 protein and a non-Chk1 protein sequence. It is not intended that the fusion proteins of the present invention be limited to any particular portion of the Chk1 portion or any particular non-Chk1 protein sequences. In preferred embodiments, the fusion the Chk1 protein portion of the fusion protein comprises at least a portion of SEQ ID NO:4. In an alternative embodiment, the non-Chk1 protein sequence comprises an affinity tag. In particularly preferred embodiment, the affinity tag comprises a histidine tract.

The present invention also provides methods for detecting Chk1 protein. In one embodiment, the method comprises the steps of providing in any order: a sample suspected of containing the Chk1 protein; an antibody capable of specifically binding to a Chk1 protein; mixing the sample and the antibody under conditions wherein the antibody can bind to the Chk1 protein; and detecting the binding. In one alternative embodiment, the sample comprises one or more cells suspected of containing Chk1 protein. In yet another embodiment, the cells contain an abnormal Chk1 protein. In a further embodiment, the cells are selected from the group consisting of human cells and murine cells.

The present invention also provides antibodies capable of recognizing at least a portion of human and/or murine Chk1 protein. In one embodiment, the present invention provides an antibody, wherein the antibody is capable of specifically binding to at least one antigenic determinant on the proteins encoded by an amino acid sequence selected from the group comprising SEQ ID NOS:3, 4, 7, 8, 9, and 10. In one preferred embodiment, the antibody is a polyclonal antibody, while in an alternative embodiment, the antibody is a monoclonal antibody.

The present invention also provides methods for producing antibodies comprising the steps of providing in any order: an antigen comprising at least a portion of Chk1 protein; and an animal having immunocompetent cells; and exposing the animal to the Chk1 protein under conditions such that the immunocompetent cells produce anti-Chk1 antibodies. In one alternative embodiment, the method further comprises the step of harvesting the antibodies. In another alternative embodiment, the antigen comprises at least a portion of Chk1 protein is a fusion protein. In yet another embodiment, the method further comprises the step of fusing the immunocompetent cells with an immortal cell line under conditions such that an hybridoma is produced.

The present invention also provides methods for detection of polynucleotides encoding human and/or murine Chk1 in biological samples. It is not intended that the method be limited to any particular sequence contained within SEQ ID NOS:1 or 2. Indeed, it is contemplated that any sequence be used in the method, including degenerate primers that are based on the sequence of chk1spChk1

The library was constructed using methods known in the art (
Primer 186: 5′-gca gtt tgc agg aca gga taa tct tct cta gga ag-3′  (SEQ ID NO:13)
Primer 415: 5′-ttg ctc cag aac ttc tg-3′  (SEQ ID NO:14)
Primer 484. 5′-tat tgg ttg act tcc ggc-3′  (SEQ ID NO:15)

By automated sequence analysis, it was determined that this clone contained the chk1 CHK1 sequence. This clone was then used in FISH analysis as known in the art, in order to determine the chromosomal location of the chk1 CHK1 gene.

The results of this analysis placed the gene at a position that is adjacent to the gene encoding ATM on chromosome 11 (i.e., at 11q23). Loss of heterozygosity at this region has been associated with a number of cancers, including breast, lung, and ovarian cancers (I. Vorechovsky et al., Cancer Res., 56:2726 [1996]; and H. Gabra et al., Cancer Res., 56:950 [1996]).

In the Northern analyses, mRNAs from human and mouse tissues were hybridized with 25 ng of labeled human or mouse cDNAs, as appropriate, overnight in 50 mM PIPES, 100 mM NaCl, 50 mM Na₂HPO₄, 1 mM EDTA, and 5% SDS, at 65° C. The blots were washed in PIPES at room temperature, followed by a high stringency wash in 0.1×SSC with 0.5% SDS, at 65° C., for 40 minutes.

The results of the Northern blot analysis (as shown in FIGS. 5A and 5B), revealed the ubiquitous expression of hChk1 hCHK1, with large amounts present in human thymus, testis, small intestine, and colon. In adult mice, mChk1 mChek1 was detected in all tissues examined, and large amounts were found in the testis, spleen and lung. In addition, mouse embryos from embryonic day 15.5 also revealed ubiquitous expression, with large amounts detected in the brain, liver, kidney, pancreas, intestines, thymus, and lung. These results were of particular interest, as testis, spleen, and thymus have also been found to express large amounts of ATM (G. Chen and E. Y. H. P. Lee, J. Biol. Chem., 271:33693 [1996]; and N. D. Lakin et al., Oncogene 13:2707 [1996]).

EXAMPLE 3

Antibodies Against Chk1

In this Example, affinity-purified antibodies to hChk1 protein (“anti-FL”) and the 15 amino acids present on the carboxy terminus of the hChk1 protein (“anti-PEP”) were produced. In these experiments, hChk1 protein was first produced in baculovirus as described below.

Recombinant baculovirus encoding glutathione S-transferase (GST) fusion proteins to hCHk1 hChk1 (GST-hChk1 hCHK1) or a to a mutation of hChk1 in which Asp at position 130 was mutated to Ala (GST-hChk1 hCHK1 (D130A)) were produced. Recombinant baculovirus encoding GST-hChk1 and GST-hChk1(D130A) (pYS71) were made by introducing an NdeI at the first ATG of the hChk1 hCHK1 open reading frame (ORF) using PCR, and subcloning the hChk1 hCHK1 cDNA as an Nde I-EcoRI fragment into pGEX2Tcs (Invitrogen INVITROGEN®) to generate pYS45. The XbaI-EcoRI fragment from pYS45 containing GST-hChk1 hCHK1 was then subcloned into pVL1393 (Invitrogen INVITROGEN®), which was cut with XbaI-EcoRI to generate pYS63.

The GST-hChk1(D130A) mutant was generated by the PCR and the XhoI-XmnI fragment containing the mutation was used to replace the wild-type fragment to generate pYS64. The hGST-Chk1(D130A) fragment from pYS64 was then subcloned into the baculovirus transfer vector using the Univector plasmid fusion strategy, as described in co-pending U.S. Patent Application Ser. No. 08/864,224, now issued as U.S. Pat. No. 5,851,808, hereby incorporated by reference.

Viruses were generated by standard methods (e.g., Baculogold BACULOGOLD®, Pharmingen Corporation, San Diego, Calif.). Recombinant GST-hChk1 protein was isolated from infected Hi5 insect cells (Invitrogen INVITROGEN®) on glutathione (GSH) agarose (Pharmacia PHARMACIA®).

The GST-hChk1 protein was then used to produce affinity-purified antibodies. In addition, antibodies directed against the carboxy-terminal 15 amino acids were produced using synthetically produced sequence. Recombinant GST-hChk1 was affinity purified from the cell lysate by chromatography on Glutathione Sepharose SEPHAROSE 4B™ (Pharmacia GE Healthcare Bio-Sciences AB LLC, Uppsala, Sweden) according to the manufacturer's instructions.

Polyclonal antibodies against the purified GST-hChk1 or the carboxy terminal amino acids were generated in New Zealand white rabbits (Bethyl Laboratories), using standard techniques (See see e.g., E. Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York [1988]). Briefly, rabbits were given an initial immunization comprising 100 μg of affinity purified GST-hChk1 or the 15 amino acid sequence in complete Freund's adjuvant (CFA). The antigen was delivered by SC injection. The animals received boosts comprising 50 μg of affinity purified GST-hChk1 or 15 amino acid sequence, in incomplete Freund's adjuvant (IFA), as appropriate, at the following intervals day 14, day 28, day 42, day 56 and day 70. Sera were collected by bleeding the rabbits from the ear vein and the sera were prepared using standard techniques (E. Harlow and D. Lane, supra, at pp. 117 and 119). The anti-hCHk1 hChk1 antibodies were referred to as “anti-FL,” while the antibodies directed against the carboxy-terminal 15 amino acids were referred to as “anti-PEP.”

Anti-PEP antibodies were purified using an affinity column that was prepared by coupling a peptide representing the carboxy-terminal 15 amino acids, at its N terminus to activated CH-Sepharose SEPHAROSE 4B™ (Pharmacia GE Healthcare) according to the manufacturer's instructions. The anti-FL antibodies were purified using an affinity column that was prepared by coupling the GST-Chk1 fusion protein from baculovirus to Affi-Gel AFFI-GEL® 10 (Biorad BIO-RAD®) according to the manufacturer's directions. The antibody concentrations were roughly determined by Bradford analyses. The antibodies were subsequently tested in titration experiments and in Western blots, to determine their titer and specificity.

Affinity purified antibodies to these hChk1 protein made in baculovirus (“anti-FL”) or to its COOH-terminal 15 amino acids (“anti-PEP”), recognized a 54-kD protein (FIG. 6) that comigrates with hChk1 expressed in baculovirus. The anti-PEP but not anti-FL signal is competed by addition of excess peptide indicating that the two sera are recognizing different hChk1 epitopes, further confirming identity of the 54-kD band as endogenous hChk1. A 70-kD protein was also specifically recognized by anti-PEP.

Antibodies directed against mChk1 were also produced and purified, using the same methods as described above for the anti-hChk1 antibodies.

mChk1 expressed from the cytomegalovirus promoter, CMV, in baby hamster kidney cells (BHK) resulted in detection of a 54-kD nuclear protein only in transfected cells using antibodies directed against the C-terminal peptide of mChk1. These (and all other transfections) were carried out as follows. Tissue culture flasks (T25) at 70-80% confluence were incubated with 3-9 μg DNA and 15-18 μl lipofectamine (Gibco BRL), in 3 ml of OptiMEMI OPTI-MEM® (Gibco BRL GIBCO®), for 5-7 hours at 37° C. The cells were washed three times with Dulbecco's PBS without calcium or magnesium, and fed with DMEM with high glucose (Gibco BRL GIBCO®) and 10% FBS (Gibco BRL GIBCO®). The cells were harvested for Western blots or FACS analyses 48 hours post transfection. The results indicated exogenous mChk1 comigrates with endogenous mChk1 from mouse lung tissue.

EXAMPLE 4

Effect of DNA Damage

To determine whether hChk1 is modified in response to DNA damage like spChk1, hChk1 protein in extracts from cells treated with ionizing radiation was examined.

In the first set of experiments, HeLa cells were synchronized with 2 mM thymidine, and treated without (−) or with (+) 10 Gy of ionizing radiation one hour after release from the block. Cells were collected in G2-M, and extracts were fractionated by 10% SDS-PAGE, and immunoblotted with anti-PEP.

In addition to the HeLa cells, Jurkat cells were treated (+IR) or not treated (−IR) with 10 Gy of ionizing radiation and incubated for two hours. Extracts from these cells were resolved in the first dimension by using isoelectric focusing (IEF), with pH 3 to 10 ampholytes, and in the second dimension on a 10% SDS-PAGE, followed by immunoblotting with anti-PEP.

hChk1 from extracts from damaged cells showed a minor but reproducible reduction in mobility compared to Chk1^Hs from untreated cells (FIG. 7). This modification was confirmed by 2-dimensional gel analysis which clearly demonstrated the generation of a more negatively charged Chk1 species 2 hours after γ-irradiation (FIG. 7). These results indicate that hChk1 may participate in transduction of the DNA damage signal like spChk1.

Indirect immunofluorescence was also conducted. In these experiments, human fibroblasts were fixed, stained with 4′6′-diamidino-2-phenylindole (DAP) to detect DNA, and were probed with affinity-purified anti-PEP, biotinylated antibody to rabbit IgG, and Texas Red streptavidin to reveal the subcellular location of the hChk1 protein. This indirect immunofluorescence revealed that hChk1 is localized to the nucleus in a punctate staining pattern, similar to that observed for ATM.

mChk1 was also tested as described above for hChk1, with the exception that it was expressed in BHK cells. These results also confirmed the nuclear localization of mChk1.

Finally, in order to test for the ability of Chk1^Hs to regulate the cell cycle, hChk1 hCHK1 or hChk1 hCHK1 (D130A) were transfected under the control of the cytomegalovirus (CMV) promoter, or the CMV vector alone into HeLa cells treated with and without 6 Gy of ionizing radiation. These transfections were accomplished as described in Example 3, above. No perturbation of the cell cycle by either kinase relative to vector alone was detected, suggesting that overproduction alone was insufficient to deregulate the system.

EXAMPLE 5

Phosphorylation of Cdk Tyrosine Phosphorylation Regulators

In this Example, the effects of phosphorylation of key regulators of Cdk tyrosine phosphorylation by chk1 Chk1 was investigated.

Tyrosine phosphorylation of Cdc2 has been implicated in cell cycle arrest in response to DNA damage and replication blocks in both S. pombe (T Enoch and P. Nurse, Cell 60 665 [1990]), and humans (P. Jin et al., J. Cell Biol., 134.963 [1996]). In S. pombe, Cdc2 mutants that cannot be phosphorylated on tyrosine display an inability to arrest the cell cycle in response to blockade of DNA replication. Although it was originally thought that the DNA damage checkpoint did not operate through tyrosine phosphorylation, tyrosine phosphorylation is apparently required for S pombe cells to arrest in response to DNA damage. While it is now clear that tyrosine phosphorylation is required for proper checkpoint control, the experiments implicating tyrosine phosphorylation in this pathway do not distinguish between a regulatory role in which tyrosine phosphorylation rates are manipulated by the checkpoint pathways, or a passive role in which tyrosine phosphorylation is required to allow cell cycle arrest, but is not the actual target of the checkpoint pathway (S. J. Elledge, Science 274: 1664 [1996]; and D. J. Lew and S. Kombluth, Curr. Opin. Cell. Biol., 8:795 [1996]).

Next, the ability of hChk1 to phosphorylate key regulators of Cdk tyrosine phosphorylation, the Cdc25 dual specificity phosphatases, hCdc25A, hCdc25B, and hCdc25C was analyzed. These regulators were chosen for several reasons. First, overproduction of hCdk4 mutants in which the inhibitory tyrosine is changed to phenylalanine abrogates G1 arrest in response to UV light (Y. Terada et al., Nature 376:358 [1995]). Secondly, the UV-sensitivity of chk1 Chk1⁻ mutants in S. pombe is suppressed by inactivating cdc25 with a Ts mutation (N. C. Walworth et al., Nature 363:368 [1993]). Finally, in S. pombe wee1mik1 mutants, DNA damage still causes a partial cell cycle delay that could be due to regulation of spCdc25 activity.

GST-hChk1 and GST-hChk1(D130A) were introduced into baculovirus, purified from baculovirus-infected insect cells as described in Example 3 above, and incubated with either GST, His₆-Cdc25C, GST-hCdc25A, hGST-Cdc25B, GST-hCdc25C, or GST-Cdc25C(200-256), and (γ³²P)ATP.

The kinase reactions contained hGST-Chk1 bound to GSH agarose and either His₆-Cdc25C, GST-Cdc25A, GST-Cdc25B, GST-Cdc25C or GST-Cdc25C(200 to 256) (i.e., amino acids 200 to 256 of Cdc25). Kinase reactions contained 1 to 3 μg of GST-hChk1 or GST-hChk1(D130A) protein on beads and soluble substrate in 20 mM Hepes (pH 7.4), 10 mM MgCl₂, 10 mM MnCl₂, 2 μM ATP and 15 μCi (γ-³²P)ATP for 30 minutes at 30° C. The proteins were resolved by SDS-PAGE (10%), and visualized by autoradiography for kinase assays (FIG. 8A), or by Coomassie staining (FIG. 8B). Less GST-Cdc25B was loaded than the other substrates (approximately ⅕ of the other substrates was loaded).

GST-Chk1 phosphorylated all three Cdc25 proteins but not GST alone (FIG. 8). Although Gst-Cdc25C^Hs co-migrated with Gst-Chk1^Hs which autophosphorylates, increased phosphorylation was observed at that position relative to that in the presence of kinase alone and phosphorylation of a Gst-Cdc25C^Hs breakdown product was visible.

Protein kinases often form complexes with their substrates. To examine this for hChk1, and the Cdc25 proteins, GST-Cdc25 proteins present on glutathione beads were incubated together with baculovirus extracts expressing His₆-tagged hChk1, and precipitated GST-hCdc25A, GST-hCdc25B, and GST-hCDC25C each specifically bound hChk1 while GST alone did not (FIG. 10). Furthermore, two other GST fusion proteins, GST-Dun1 and GST-Skp1, all failed to bind hChk1. These results indicate that Cdc25 can form complexes with hChk1.

To determine the site on Cdc25C that is phosphorylated by hChk1, the kinase reactions were carried out in a buffer consisting of 50 mM Tris (pH 7.4), 10 mM MgCl₂, 10 μM ATP, 1 mM DTT and 10 μCi (γ-³²P)ATP. The proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and visualized by autoradiography. The nitrocellulose membrane containing His-Cdc25C was excised, blocked with 0.5% polyvinylpyrrolidone (PVP-40) in 100 mM acetic acid for 30 minutes at 37° C., washed six times with water, and digested with TPCK trypsin (Worthington Biochemical Corporation, Lakewood, N.J.) at a final concentration of 30 mg/ml, in 0.1 M NH₄CO₃ (pH 8.0). Further digestion on selected HPLC fractions was performed with 2 units of proline specific endopeptidase (ICN VALEANT®) in 0.1M sodium phosphate, 5 mM EDTA (pH 7.4), at 37° C. for 16 hours. Samples were acidified in 1% trifluoroacetic acid (TFA) and loaded onto a Vydac VYDAC® C18 column (25 cm×0.46 cm inner diameter, registered to Alltech Associates, Inc., Columbia, Md.). Reverse phase HPLC was performed at 37° C. Reactions were loaded in 0.1% TFA (Buffer A) and eluted with a gradient from 0 to 60% Buffer B (90% acetonitrile, 0.095% TFA). Fractions were collected at 0.5 minutes intervals up to 90 minutes, and counted for radioactivity. Selected fractions were immobilized on Sequenlon-AA membrane discs (Millipore Corporation, Billerica, Mass.) for NH₂-terminal sequencing. Manual Edman degradation was done as known in the art (See see, J. E. Rodwell et al, J. Biol. Chem., 266:7549 [1991]; and S. Sullivan, and T. W. Wong, Anal. Biochem., 197: 65 [1991]) with a coupling and cleavage temperature of 55° C.

To establish the significance of the Cdc25 phosphorylation, the site of Chk1^Hs phosphorylation on Cdc25C was mapped. Ser 216 is the main site of phosphorylation of Cdc25C^HS in vivo. hChk1 phosphorylated a 56 amino acid region of the hCdc25C protein fused to GST, but not GST alone (FIG. 8). This 56 amino acid motif contains 4 possible sites of phosphorylation. peptide analysis of proteolytic fragments of full length His₆-hCdc25 C phosphorylated with GST-hChk1 revealed a single phosphorylated tryptic peptide by high pressure liquid chromatography. Edman degradation of this peptide indicated release of radioactivity in the third cycle (FIG. 12B). FIG. 12A shows the radioactivity measured from column fractions obtained during reverse phase HPLC. Further degradation of this tryptic fragment with proline endopeptidase resulted in a peptide that released radioactivity in the first cycle. Serine 216 is the only site on Cdc25C^Hs consistent with this phosphorylation pattern (FIG. 11), as amino acids inclusive of and surrounding Serine²¹⁶ contain amino-terminal trypsin and proline endopeptidase cleavage sites.

In addition, GST-hChk1 purified from baculovirus was incubated with either GST-hCdc25C(200-256) or GST-hCdc25C(200-256)(S216A), and (γ-³²P)ATP, using the same methods as described above. The results are shown in FIG. 9A). In addition, hChk1-His₆ purified from baculovirus was incubated with either GST-hCdc25C (lane 5, FIG. 9B), or GST-hCdc25c(S216A) and (γ-³²P)ATP. Proteins were resolved and visualized as described above. As shown in FIG. 9, there was clear phosphorylation of GST-hCdc25C. A catalytically inactive mutant (GST-hChk1(D130A)(k−) failed to phosphorylate itself or any of the Cdc25 proteins (See, FIG. 9).

To confirm this, the Cdc25C S216A mutation in Gst-Cdc25C and Cdc25C(200-256) were constructed. Both were found to be poor substrates for hChk1 confirming S216 as the site phosphorylation (FIG. 11). S216 has also been reported to be phosphorylated by spChk1, demonstrating phylogenetic conservation of this regulatory relationship.

EXAMPLE 6

Production of Monoclonal Antibodies

The antibodies of the present invention may be monoclonal or polyclonal. Thus, it is within the scope of this invention to include other (e.g., second antibodies) (monoclonal or polyclonal) directed against or similar to the first antibodies discussed above. It is contemplated that these antibodies will find use in detection assays. Both the first and second antibodies may be used in the detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of human or murine Chk1.

The production and use of monoclonal antibodies in an immunoassay is an alternative method to that described in Example 3. Monoclonals provide some advantages because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See e.g., Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz [1981]; Kohler and Milstein, Nature 256: 495-499 [1975]; Eur. J Immunol., 6: 511-519, [1976]).

Unlike preparation of polyclonal sera, the choice of animal is dependent on the availability of appropriate immortal lines capable of fusing with lymphocytes. Mouse and rat have been the animals of choice in hybridoma technology and are preferably used. Humans can also be utilized as sources for sensitized lymphocytes if appropriate immortalized human (or nonhuman) cell lines are available. For the purpose of the present invention, the animal of choice may be injected with an antigenic amount, for example, from about 0.1 mg to about 20 mg of the enzyme or protein or antigenic parts thereof. Usually the injecting material is emulsified in Freund's complete adjuvant. Boosting injections may also be required. The detection of antibody production can be carried out by testing the antisera with appropriately labell ed antigen. Lymphocytes can be obtained by removing the spleen of lymph nodes of sensitized animals in a sterile fashion and carrying out fusion. Alternatively, lymphocytes can be stimulated or immunized in vitro, as described, for example, in Reading, J. Immunol. Meth., 53: 261-291 [1982]) .

A number of cell lines suitable for fusion have been developed and the choice of any particular line for hybridization protocols is directed by any one of a number of criteria such as speed, uniformity of growth characteristics, deficiency of its metabolism for a component of the growth medium, and potential for good fusion frequency.

Intraspecies hybrids, particularly between like strains, work better than interspecies fusions. Several cell lines are available, including mutants selected for the loss of ability to secrete myeloma immunoglobulin.

Cell fusion can be induced either by virus, such as Epstein-Barr or Sendai virus, or polyethylene glycol. Polyethylene (PEG) is the most efficacious agent for the fusion of mammalian somatic cells. PEG itself may be toxic for cells and various concentrations should be tested for effects on viability before attempting fusion. The molecular weight range of PEG may be varied from 1000 to 6000. It gives best results when diluted to from about 20% to about 70% (w/w) in saline or serum-free medium. Exposure to PEG at 37° C. for about 30 seconds is preferred in the present case, utilizing murine cells. Extremes of temperature (i.e., about 45° C.) are avoided, and preincubation of each component of the fusion system at 37° C. prior to fusion can be useful. The ratio between lymphocytes and malignant cells is optimized to avoid cell fusion among spleen cells and a range of from about 1.1 to about 1:10 is commonly used.

The successfully fused cells can be separated from the myeloma line by any technique known by the art. The most common and preferred method is to choose a malignant line which is Hypoxthanine Guanine Phosphoribosyl Transferase (HGPRT) deficient, which will not grow in an aminopterin-containing medium used to allow only growth of hybrids and which is generally composed of hypoxthanine, 1×10₋₄M 1×10⁻⁴ M, aminopterin 1×10⁻⁵ M, and thymidine 3×10⁻⁵ M, commonly known as the HAT medium. The fusion mixture can be grown in the HAT-containing culture medium immediately after the fusion 24 hours later. The feeding schedules usually entail maintenance in HAT medium for two weeks and then feeding with either regular culture medium or hypoxthanine, thymidine-containing medium.

The growing colonies are then tested for the presence of antibodies that recognize the antigenic preparation. Detection of hybridoma antibodies can be performed using an assay where the antigen is bound to a solid support and allowed to react to hybridoma supernatants containing putative antibodies. The presence of antibodies may be detected by “sandwich” techniques using a variety of indicators. Most of the common methods are sufficiently sensitive for use in the range of antibody concentrations secreted during hybrid growth.

Cloning of hybrids can be carried out after 21-23 days of cell growth in selected medium. Cloning can be preformed by cell limiting dilution in fluid phase or by directly selecting single cells growing in semi-solid agarose. For limiting dilution, cell suspension are diluted serially to yield a statistical probability of having only one cell per well. For the agarose technique, hybrids are seeded in a semi-solid upper layer, over a lower layer containing feeder cells. The colonies from the upper layer may be picked up and eventually transferred to wells.

Antibody-secreting hybrids can be grown in various tissue culture flasks, yielding supernatants with variable concentrations of antibodies. In order to obtain higher concentrations, hybrids may be transferred into animals to obtain inflammatory ascites. Antibody-containing ascites can be harvested 8-12 days after intraperitoneal injection. The ascites contain a higher concentration of antibodies but include both monoclonals and immunoglobulins from the inflammatory ascites. Antibody purification may then be achieved by, for example, affinity chromatography.

Antibodies produced by these methods can then be used in immunoassay methods to detect human or murine Chk1. Such methods include, but are not limited to ELISA (enzyme-linked immunosorbent assay), IFA (immunofluorescence assay), or RIA (radioimmunoassay).

From the above it should be clear that the present invention provides gene sequences encoding mammalian checkpoint genes and proteins useful as probes for a tumors and other malignancies, as well as growth and/or development deficiencies.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. A purified human protein, wherein the protein is a polypeptide having checkpoint kinase activity and encoded by the nucleotide sequence of SEQ ID NO: 3.

2. A purified human protein comprising the amino acid sequence set forth in having checkpoint kinase activity, wherein the protein comprises SEQ ID NO: 1.

3. A fusion protein comprising a portion of at least 15 sequential amino acids of the a carboxy-terminus of the CHK1 protein of claim 2, sequence of SEQ ID NO:1 and a non-CHk1 Chk1 protein sequence .

4. The fusion protein of claim 3, wherein said non-Chk1 protein sequence comprises is combined with an affinity tag for purification of the fusion protein.

5. The fusion protein of claim 4, wherein said affinity tag comprises is a histidine tag.

6. A purified Chk1 protein encoded by the nucleotide sequence of claim 5.

7. The fusion protein of either claim 3 or claim 6, wherein the non-Chk1 protein is glutathione S-I transferase.