AU7366900A - Methods and compositions for the screening of cell cycle modulators - Google Patents

Description

WO 01/20034 PCTIUSOO/24838 METHODS AND COMPOSITIONS FOR THE SCREENING OF CELL CYCLE MODULATORS Related Information 5 The contents of the patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties. Background of the Invention Key transition points in the eukaryotic cell cycle are regulated by the activity of 10 cyclin dependent kinases (cdks) (Nurse, P., (1990) Nature 344(6266):503-508 for review see: Fisher, R.P. (1997) Curr Opin Genet Dev 7(1):32-38). Activity of these molecules is regulated by many mechanisms including phosphorylation and protein protein interactions (Lew, et al., (1996) Curr Opin Cell Biol 8:795-804). For example, phosphorylation of Cdk by Cdk7 (on e.g., threonine residue 160) activates Cdk activity 15 whereas phosphorylation by MytI and Weel (on Thr-14 and Tyr-15) is inhibitory (Gould, K.L., et al. (1989) Nature 42(6245):39-45; Krek, W., et al. (1991) EMBO J 10(2):305-16; Russell, P., et al., (1987) Cell 49(4):559-567). Moreover, Cdk activity can be regulated by the dephosphorylation of these residues by, for example, a Cdc25 dual-specificity protein phosphatase which is a member of a group of highly related dual 20 specificity phosphatases that promote cell cycle phase transitions (Galaktionov, et al., (1991) Cell 67:1181-1194; Millar, J.B., et al., (1991) EMBO J 10(13):4301-4309). Cdc25 phosphatases are expressed in all eukaryotes. A single form of Cdc25 is expressed in yeast (Russell, P., et al. (1986) Cell 45(1):145-153) and two forms are present in Drosophila (Edgar, B.A., et al. (1989) Cell 57(1):177-187; Alphey, L., et al., 25 (1992) Cell 69, 977-988). In mammals, three forms of Cdc25 (A, B and C) are present and are encoded by three highly related but distinct genes (reviewed by Draetta, et al., (1997) Biochem etBiophysActa 1332:M53-M63). Recently a fourth form of Cdc25 was reported in C. elegans raising the possibility that other forms may exist in mammalian cells (Ashcrot, N.R., et al., (1998) Gene 214:59-66). A crystal structure has been 30 deduced for human Cdc25A (Fauman, E. B., et al., (1998) Cell 93, 617-625) WO 01/20034 PCT/USOO/24838 -2 Separate but perhaps over lapping functions have been assigned to each form of Cdc25. Cdc25A has been proposed to mediate transition from GI to S phase (Jinno, S., et al., (1994) EMBO Journal 13, 1549-1556). The role of Cdc25B has been cast both at the Gl/S and G2M transitions (Sebastian et al. (1993) Proc Natl Acad Sci USA 90(8):3521 5 3524; Galaktionov, K., et al., (1995) Genes & Development 9, 1046-1058; Gabrielli, B.G., et al., (1996) J Cell Sci 109(Pt 5):1081-1093; Garner-Hamrick, et al., (1998) Int J Cancer 76:720-728) and Cdc25C is required for the onset of mitosis (Karlsson C., et al., (1999) J Cell Biol 146(3):573-584). Based on gene disruption studies, Cdc25 phosphatases clearly play a role in cell 10 cycle progression in yeast and Drosophila (Russell, P., et al. (1986) Cell 45(l):145-153; Edgar, B.A., et al. (1989) Cell 57(1):177-187; Alphey, L., et al., (1992) Cell 69, 977 988). In mammalian cells, at least one report implicates Cdc25A in regulating GI arrest due to DNA damage (Terada, Y., et al., (1995) Nature 376, 358-362). In addition, Cdc25A has been reported to play a role in regulating cell cycle progression in response 15 to serum factors. For example, TGFp was proposed to cause GI arrest in certain cell types by inhibiting Cdc25A expression (lavarone, A., et al, (1999) Mol Cell Biol 19, 916-922; Hoffman, I., et al., (1994) EMBO Journal 13, 4302-4310). Cdc25A has also been proposed to lie at the end of signal transduction pathways activated by mitogens (Galaktionov, K., et al., (1996) Nature 382, 511-517; Galaktionov, K., et al., (1995) 20 Genes & Development 9:1046-1058). In the mouse, Cdc25A and B are expressed in overlapping but distinct patterns in adult animals as well as in embryos (Wickramasinghe, D., et al., (1995) Development 121, 2047-2056; Kakizuka, A., et al., (1992) Genes & Development 6, 578-590). In adult mice, Cdc25A is expressed most abundantly in testes and kidney and is barely 25 detectable in lung and spleen (Wickramasinghe, D., et al., (1995) Development 121, 2047-2056). In the adult rat, Cdc25B is expressed most abundantly in spleen and lung but not detectable in kidney (Kakizuka, A., et al., (1992) Genes & Development 6, 578 590). In adult mice, Cdc25C is expressed most abundantly in thymus but was not detected in lung or kidney (Nargi, J. L., et al., (1994) Immunogenetics 39, 99-108). 30 Consistent with their proposed role as cell cycle regulators, over-expression of Cdc25A and B, but not C, has been reported in breast cancer (Galaktionov, K., et al., (1995) Genes & Development 9, 1046-1058), non-Hodgkin lymphomas (Hernindez, S., WO 01/20034 PCT/USOO/24838 -3 et al., (1998) Cancer Research 58, 1762-1767), in head and neck cancers (Gasparotto, D., et al., (1997) Cancer Research 57, 2366-2368), and in human gastric carcinomas (Kudo, Y., et al., (1997) Japanese Journal of Cancer Research 88, 947-952). Moreover, it has been reported that Cdc25A and B, but not C, can cooperate with the activated ras 5 oncogene to immortalize mouse embryo fibroblasts (Galaktionov, et al., (1995) Science 269:1575-1577). The mechanism by which Cdc25 polypeptides regulate the cell cycle is still incompletely understood. Moreover, current methods for identifying modulators of Cdc25 activity involve assays that are directed to screening for compounds that alter 10 either, 1) Cdc25 phosphatase activity, or, 2) the ability of a Cdc25 polypeptide to cause a phenotypic effect such as, e.g., apoptosis. Examples of techniques for identifying modulators of Cdc25 phosphatase activity are presented in, e.g., USPN 5,695,950, where potential inhibitor compounds are incubated with Cdc25 and a substrate, and a change in the ability of Cdc25 to 15 dephosphorylate the substrate is taken as indicative of the test compound as modulating Cdc25 phosphatase activity. Similarly, in USPN 5,294,538, an assay is presented for screening anti-mitotic compounds using a Cdc25 phosphatase and test substrate, e.g., p nitrophenyl, and Cdc25 phosphatase activity on the substrate in the presence or absence of a candidate compound is determined. And, in USPN 5,856,506 and USPN 5,700,821, 20 compounds for inhibiting the phosphatase activity of protein phosphatases such as Cdc25A and Cdc25B are presented. Examples of techniques for assaying the ability of a Cdc25 polypeptide to cause a phenotypic effect such as, e.g., apoptosis, are presented in USPN 5,443,962, where an assay for identifying an inhibitor of Cdc25 phosphatase causing apoptosis is scored 25 based on the ability of the inhibitor to cause the cell to proliferate. Another phenotypic assay is presented in USPN 5,861,249, where modulators of Cdc25 are identified based on their ability to alter Cdc25-induced apoptosis as compared to a control. In summary, all of the foregoing assays are directed to screening compounds that alter, either phosphatase activity on a substrate, e.g., an artificial substrate such as p 30 nitrophenyl, or the ability of the modulator to alter a Cdc25-related change of a phenotypic effect such as, e.g., apoptosis.

WO 01/20034 PCT/USOO/24838 -4 Accordingly, these assays have several limitations. The assays either look at phosphatase activity in isolation of any downstream or biological effect, or, are designed to begin with cells that are severely compromised in their growth and yield a qualitative result. Thus, candidate compounds that may function in a phosphatase assay may not 5 have been assayed for any downstream or biological efficacy. In addition, potentially useful compounds that are being assayed at levels or in a form that is incompatible with cell growth, may be erroneously dismissed as ineffective. Accordingly, a need for improved assays for identifying modulators of Cdc25 exists. 10 Summary of the Invention The invention solves the foregoing problems by providing a novel assay for screening modulators of Cdc25 that relies on a robust cell culture system that can identify a candidate modulator by the appearance of a return or "rescue" of a strong 15 reporter gene signal. The assay employs a catalytically active form of a Cdc25 that can repress a selected promoter driving strong gene expression. In parallel, a control is performed using a catalytically inactive form of Cdc25 that does not repress promoter activity but allows for a strong baseline signal to be established. This important control allows for determining if a particular test compound 20 is inhibiting promoter activity independently of Cdc25. If the control signal is essentially unaffected, than the signal measured from the test reaction containing the catalytically active form of a Cdc25 can be accurately interpreted. Moreover, candidate modulators of Cdc25, e.g., inhibitors of Cdc25-mediated gene regulation, will cause a strong signal to appear. 25 Accordingly, the invention has several advantages which include, but are not limited to, the following: - provides an assay that measures the appearance of a strong signal from a background of little or no signal; - provides an assay that measures the appearance of a quantifiable signal and not 30 a qualitative signal involving variable cell growth or other biological effect; WO 01/20034 PCT/USOO/24838 -5 - provides an assay that contains a control that can accurately identify compounds that are false positives (e.g., compounds that rescue the signal but also increase the signal in the test reaction) or false negatives (e.g., compounds that produce no signal but also lower the control signal, e.g., cytotoxic compounds) and this insures 5 that inappropriate compounds are not further investigated and that candidate compounds are not erroneously dismissed; - provides an assay that measures Cdc25-mediated gene regulation, e.g., at the level of promoter control, and provides promoters that are mediated by Cdc25 (e.g., the p21/WAF promoter; SV40 promoter) and control promoters that are not (e.g., the globin 10 promoter); and - provides Cdc25A genomic and cDNA nucleic acids, polypeptides, and transgenic animals having a Cdc25 gene disruption for extending determinations made using, e.g., the above assay for studies relating to Cdc25 autoregulation and/or studies requiring cells or an animal model with lowered or absent levels of a Cdc25 polypeptide. 15 Accordingly, in one aspect, the invention provides a method for identifying an modulator of Cdc25 activity by providing a cell having a recombinant Cdc25 phosphatase gene where the expression of the gene alters the transcription of a selected gene. The method further includes contacting the test cell with a compound under conditions where the recombinant Cdc25 phosphatase gene is expressed and alters the 20 transcription of a selected gene and determining the amount of transcription of a selected gene in the test cell as compared to the amount of transcription of the selected gene in the absence of the compound where a statistically significant change in the amount of transcription of the selected gene is indicative of the compound being a modulator of Cdc25-mediated transcription. 25 In one embodiment, the assay further includes comparing the change in transcription measured in the above aspect in comparison with the transcription measured from a control test cell having a recombinant catalytically inactive Cdc25 phosphatase under conditions where the catalytically inactive Cdc25 phosphatase is expressed and does not substantially alter the transcription of the selected gene and 30 determining the amount of transcription of the selected gene in the control test cell in the absence of the test compound as compared to the amount of transcription of the selected gene in the presence of the compound where a statistically significant change in the WO 01/20034 PCTIUSOO/24838 -6 amount of transcription of the selected gene is indicative of the compound as a modulator of transcription independent of Cdc25. In one embodiment, the selected gene is a eukaryotic gene that contains, or is operably linked, to a eukaryotic promoter element. In a related embodiment, the 5 selected gene is p21/WAF, pGK, or Cdc25. In a related embodiment, the selected gene contains a reporter gene, preferably, luciferase. In a related embodiment, the reporter gene is controlled by a cellular promoter such as the p21 /WAF promoter, pGK promoter, or a Cdc25 promoter. In another embodiment, the reporter gene is controlled by a viral promoter, 10 preferably the SV40 promoter. In another embodiment, the recombinant Cdc25 phosphatase gene of the above aspect encodes a mammalian Cdc25 phosphatase, preferably a mouse Cdc25 phosphatase, more preferably a human Cdc25 phosphatase. In a related embodiment, the Cdc25 phosphatase, preferably a human phosphatase, is selected from the group 15 consisting of Cdc25A, Cdc25B, and Cdc25C. In even another embodiment, the above aspect employs a test cell that is a mammalian cell, preferably a murine cell, and more preferably a human cell. In a yet another embodiment, the method includes determining transcription by measuring reporter gene activity, preferably luciferase activity. 20 In preferred embodiment, a statistically significant increase in the amount of transcription determined, indicates that the test compound is an inhibitor of Cdc25 activity. In another preferred embodiment, a statistically significant decrease in the amount of transcription determined, indicates the test compound is an activator of Cdc25 25 activity. Other features and advantages of the invention will be apparent from the following detailed description and claims. Brief Description of the Drawings 30 Figure 1 shows the cDNA sequence and predicted amino acid sequence of murine Cdc25A.

WO 01/20034 PCT/USOO/24838 -7 Figure 2 shows in a schematic of the probes used for SI nuclease protection analysis (Panel A). Panel B depicts an autoradiograph of an SI nuclease protection analysis. Genomic fragments were isolated and 5' end-labeled at the indicated restriction sites. These were hybridized to RNA isolated from 129sv mouse ES cells as described 5 herein. The probe labeled at the Ncol site gave rise to a protected fragment of 420 nucleotides (nt) (700 nt undigested) whereas the probe labeled at the NotI site gave rise to a product of 260 nt (1100 nt undigested). Probes hybridized to 50 ptg yeast tRNA did not give rise to protections. 10 Figure 3 is a histogram of Cdc25A promoter activity. Mammalian 293 kidney cells (Panel A) and H460 lung cells (Panel B) were transfected in triplicate with the indicated reporter construct. Shown is the average of each triplicate transfection. Error bars indicate the standard error of the mean and the Y axis scales have been adjusted to reflect the difference in transfection efficiency of the two cell lines. 15 Figure 4 is a histogram indicating the auto-regulatory activity of Cdc25A phosphatase. Mammalian 293T cells were co-transfected with the indicated Cdc25A expression vector or an empty vector (pcDNA, Invitrogen) and 1.0 pg of the indicated luciferase (luc) reporter gene construct. Cells were harvested 48 hours later and assayed for luciferase activity. Error bars indicate the standard error of the mean; for Cdc25A luc 20 n=8, for p21/WAF luc n=7 and for P Globin n=3. The lower panel depicts a similar analysis performed on the viral promoter SV40 and the eukaryotic promoter pGK. Figure 5 shows an autoradiograph of Cdc25A RNA levels during the cell cycle. RNA samples were isolated at the indicated times before or after release from a cell 25 synchronization step (double thymidine block) and analyzed by Northern blot. Replicate blots were probed with a Cdc25A cDNA (Panel A) or ribosomal protein S14 cDNA (Panel B), quantitated using a phosphoimager, and values were normalized (Panel C). Figure 6 shows a schematic of two Cdc25A targeting vectors with the frequency 30 of homologous recombination achieved indicated. The conditional targeting vector contains the tNT cassette inserted into the NotI site (+260) in the untranslated leader sequence of the Cdc25A gene. This tNT cassette contains the coding sequences of the WO 01/20034 PCTIUSOO/24838 tTA gene, a phosphoglycerate kinase gene promoter regulated neomycin (G418) resistance gene and the Tet-o-7 tetracycline responsive promoter (Gossen, M., et al., (1992) Proc Natl Acad Sci USA 89(12):5547-555 1). The Tet responsive promoter is fused (at +62) to the NotI site in the untranslated leader sequence of the Cdc25A gene. 5 Also contained within the cassette are three polyadenylation sites and stop codons in all three reading frames. tTA driven, tetracycline dependent regulation of this tet responsive promoter can be demonstrated. The conventional targeting vector was constructed by removing a 2396 bp XcmI fragment disrupting exons 1, 2 and 3 and replacing it with a PvuI-BsteII fragment containing the pGK regulated G418 resistance gene derived from 10 pPNT. After selection for G418 resistance, EL1 ES cells electroporated with these vectors were expanded and analyzed by Southern blot for homologous recombination. Figure 7 shows a schematic of the genomic locus of murine Cdc25A and targeting vector. Neomycin transferase (Neor) contained within the tNT cassette and 15 inserted at the NotI site in exon 1, was used for positive selection. The 1.3 kb 5' and 3.2 kb 3' homology regions of murine genomic sequences are represented by closed boxes. The P1 and P2 probes used in Southern blot analysis are indicated. A Southern blot analysis to identify heterozygous ES cell DNA is shown (Panel B) where BglII digests probed with P1 detects a 7.5kb wild type band and 11.5 kb mutant band (Panel B, Upper 20 Panel) and EcoRl digests probed with P2 detects a 6.5 and a 9.5 kb wild type and mutant band respectively (Panel B, Lower Panel). Figure 8 shows photomicrographs of a histological analysis of Cdc25A heterozygous intercross embryos (A-J). Embryos at E6.5, from a maternal uterus, were 25 fixed in formaldehyde, sectioned serially and stained with hemotoxylin and eosin. All eight embryos (A-J) at E6.5 appeared healthy and displayed normal post-implantation development to the early primitive streak stage (K-T; 1 OOX) embryos (R-T) were disorganized and, in contrast to the adjacent normal embryos, had not developed to the late primitive streak stage (K-Q; 40X). 30 WO 01/20034 PCTIUS0O/24838 -9 Figure 9 shows photomicrographs of an in situ hybridization analysis of heterozygous intercross embryos at E7.5 (A-F). Darkfield and partial brightfield images overlapped of adjacent sections probed with Cdc25A (C, E) and Cdc25B (D, F). Control sense probes for Cdc25A (A, A') and Cdc25B (B, B') are indicated. Digitized darkfield 5 images of previous panel highlighting areas of hybridization to Cdc25A and Cdc25B probes as in panel I are shown in panels A'-F'. Arrows indicate position of robust Cdc25B hybridization to extra-embryonic mesodermal cells in D, D', F and F'. Figure 10 shows the complete sequence of the murine Cdc25A genomic locus. 10 Detailed Description of the Invention In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. 15 Definitions As used herein the term "Cdc25" is intended to include any art recognized cell division control 25 gene and/or corresponding polypeptide expressed therefrom. The above term is also intended to refer to related Cdc25 genes and/or polypeptides, e.g., 20 Cdc25A, Cdc25B, and Cdc25C, unless indicated otherwise. The term "Cdc25 activity" is intended to include any activity attributable to the Cdc25 polypeptide including direct catalytic activity, i.e., phosphatase activity, protein/protein interactions, and/or indirect activity such as, transcriptional modulation. The term "selected gene" is intended to include any eukaryotic gene or gene 25 capable of being transcriptionally regulated in a eukaryotic cell or cell lysate, such as, e.g., a cellular gene or a viral gene. Accordingly, the selected gene, unless otherwise noted, is typically operably linked to at least a minimal promoter and other regulatory elements, e.g., introns, termination sequences, polyadenylation sequences, etc., necessary for transcription of the gene. Accordingly, the selected gene may be a cellular gene in a 30 natural context, e.g., encoded on a chromosome or encoded on, e.g., a plasmid.

WO 01/20034 PCT/USOO/24838 - 10 The term "transcription" is intended to include any measurable transcription that occurs when the genetic information of a DNA molecule is transferred to a molecule of a messenger RNA (mRNA). The transcription may take place in a cell or in a cell lysate. In addition, levels of transcription may be measured directly as a function of, e.g., RNA 5 transcript production or indirectly as, e.g., a function of a resultant polypeptide being produced from the RNA transcript. The term is also intended to include an indirect determination of transcription by measuring, e.g., a modulation in polypeptide occupancy of a gene element, e.g., a promoter element, using, e.g., DNAse footprinting or an electrophoretic mobility shift assay (EMSA). 10 The term "statistically significant change" is intended to include any reproducible change in Cdc25 activity that is measurable with a minimum of statistical significance. Typically a change of 10%, more preferably 20%, and more preferably from 30% - 50%, and most preferably, 100%, 200%, 300%, or more, is considered a significant change. The term "eukaryotic promoter element" is intended to include any partial 15 promoter sequence which by itself is not capable of initiating normal transcription but has been determined to contribute to the activity of the overall activity of the promoter. The term "reporter gene" is intended to include any heterologous nucleotide sequence that encodes a gene product that can be conveniently assayed and includes, for example, luciferase, chloramphenicol acetyltransferase, green fluorescent protein, etc. 20 The term "cellular promoter" is intended to include a DNA sequence generally described as the 5' region of a eukaryotic gene, located proximal to the start codon. The transcription of an adjacent gene(s) is initiated at the promoter region. The term "viral promoter" is intended to include any promoter element determined to regulate the transcription of a viral polypeptide, or, when operably linked 25 to a heterologous gene, regulate the transcription of the heterologous gene in a eukaryotic cell or cell extract. Typical viral promoters intended to be encompassed by the invention include promoters derived from, e.g., SV40, adenovirus, CMV, herpesvirus, HIV, papillomavirus, AAV, etc. The term "cell" is intended to include any eukaryotic cell such as yeast cells, plant 30 cells, fungal cells, insect cells, e.g., Schneider and sF9 cells, mammalian cells, e.g., HeLa cells (human), NIH3T3 (murine), RK13 (rabbit) cells, embryonic stem cells (e.g., D3 and WO 01/20034 PCT/USOO/24838 - 11 J1), and cell types such as hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, and epithelial cells. The term "catalytically inactive Cdc25" is intended to include a Cdc25, such as, e.g., Cdc25A, that has reduced or absent levels of phosphatase activity as compared to a 5 corresponding wild type Cdc25 polypeptide. The term "test cell" is intended to include a eukaryotic cell having a catalytically active form of a Cdc25 polypeptide, e.g., a mammalian Cdc25A, B, or C. The term "control test cell" is intended to include a eukaryotic cell having a catalytically inactive form of a Cdc25 polypeptide, e.g., a mammalian Cdc25A, B, or C 10 that has reduced or absent levels of phosphatase activity as compared to a corresponding wild type Cdc25 polypeptide. Methods and Compositions The present invention will be described in detail as methods and nucleic acids, 15 nucleic acid constructs, cells containing such nucleic acids, transgenic animals, and cells derived therefrom for screening compositions (including, e.g., small molecules, peptides, polypeptides, and genes) that modulate Cdc25 (e.g., mammalian Cdc25A, Cdc25B, or Cdc25C, or a combination thereof) transcription; Cdc25 activity (e.g., phosphatase activity, protein/protein interactions); Cdc25-mediated gene regulation (including, e.g., 20 promoter repression), Cdc25-mediated cell cycle activity (e.g., apoptosis, proliferation), and Cdc25 pathways in general. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology, cell culture, and animal husbandry, which are within the skill of the 25 art and are explained fully in the literature. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); DNA Cloning, Vols. 1 and 2, (D.N. Glover, Ed. 1985); Oligonucleotide Synthesis (M.J. Gait, Ed. 1984); Nucleic Acid Hybridization (B.D. Hames and S.J. Higgins, Eds. 1984); the series Methods In Enzymology (Academic Press, Inc.), particularly Vol. 154 and Vol. 155 (Wu 30 and Grossman, Eds.; Large-Scale Mammalian Cell Culture Technology, Lubiniecki, A., Ed., Marcel Dekker, Pub., (1990); Molecular and Cell Biology of Yeasts, Yarranton et al., Ed., Van Nostrand Reinhold, Pub., (1989); Yeast Physiology and Biotechnology, WO 01/20034 PCT/USOO/24838 - 12 Walker, G., John Wiley & Sons, Pub., (1998); Baculovirus Expression Protocols, Richardson, C., Ed., Humana Press, Pub., (1998); Methods in Plant Molecular Biology: A Laboratory Course Manual, Maliga, P., Ed., C.S.H.L. Press, Pub., (1995); and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons 5 (1992)). Other features of the invention will be apparent from the following examples which should not be construed as limiting. EXEMPLIFICA TION 10 Throughout the examples, unless otherwise indicated, the following materials and methods were used unless otherwise stated. Materials and Methods 15 Cell Synchronization and Northern Blotting - Approximately 2 x 106 3T3 cells were synchronized at the Gi/S boundary by double thymidine block protocol (Pagano, M. (ed.) Cell Cycle - Materials and Methods. New York: Springer-Verlag, 1995). Cells were harvested at 0, 3, 6, 9 and 12 hours post release. A fraction of the cells were fixed, propidium iodine stained and FACS analyzed to determine cell cycle stages. Total RNA 20 from the remaining cells was isolated from 175cm 2 flasks of synchronized 3T3 cells utilizing RNAgents Total RNA Isolation System (Promega) according to the manufacturer's protocol. Twenty micrograms of total RNA per time point and 5.5ug 0.5 - 9 kb RNA marker (New England Biolabs) were electrophoresed on 1.2% agarose/formaldehyde gel and transferred to Nytran nylon membrane (Schleicher and 25 Schuell) in 1oX SSC. Prehybridization and hybridization in 5X SSPE, lOX Denhardt's, 50% formamide, 100 tg/ml salmon sperm DNA and 2% SDS was carried out at 42'C for 20 hours. The probe was a 1.7 kb C-terminal fragment of the murine Cdc25A cDNA labeled with [alpha - 32 p] using random primer method (Stratagene). After a 20 hour hybridization period, blots were washed in 2X SSC, 0.05% SDS at room temperature. A 30 second wash at 50'C in 0.1X SSC, 0.1% SDS followed. This blot was reprobed with an [alpha - 32 P] labeled S14 cDNA PCR product using the above hybridization and washing conditions, as a loading control.

WO 01/20034 PCTIUSOO/24838 - 13 Luciferase Assay - Mammalian 293T cells grown in 6-well dishes at 1 x 105 cells/well were co-transfected with 1 p.g/well effector DNA (active and inactive Cdc25A) and I tg/well reporter DNA (Cdc25A luciferase, p21 luciferase, and gamma globin) using 3 pl/well FuGENE (Boehringer-Mannheim) in 100 ptl/well serum-free medium. Forty 5 eight hours post-transfection, the cells were harvested and lysed using Promega's Luciferase Assay System. Cells were lysed in 250 pl Cell Culture Lysis Reagent and stored at -80'C until needed for assaying. In a 96-well plate, 25 Pl Luciferase Assay Reagent (luciferin) was added to 25 pl thawed cell extract. The plate was then read immediately by the luminometer. 10 Si Nuclease Analysis - An amount of 1.5 fmoles (approximately 9x10 6 cpm) of 5' end labeled probe was hybridized to 50 Vtg total RNA isolated from 129sv mouse ES cells (51 C, 80% formamide) and digested with 150 units of S1 nuclease for 1 hr at 31 C. The products of the SI nuclease digestions were fractionated on a 6% polyacrylamide gels 15 containing 8M urea and visualized by autoradiography. Immunoblot Analysis - Extracts of 293T cells transfected with various Cdc25A plasmids were resolved by electrophoresis, transferred to nitrocellulose and probed with rabbit polyclonal anti-Human Cdc25A phosphatase (1 pig/ml, Upstate Biotechnology). The 20 proteins were visualized using a donkey, anti-rabbit, secondary antibody conjugated to HRP and a chemiluminescence detection system (SuperSignal from Pierce). EXAMPLE 1 CHARACTERIZATION OF THE MURINE Cdc25A GENE 25 In this example, the characterization of the genomic locus, cDNA sequence, and amino acid sequence of murine Cdc25A, is described. Sequence and Structure of the Mouse Cdc25A Gene The complete genomic sequence of murine Cdc25A (Fig. 10) and the 30 transcription unit was determined by sequencing genomic DNA, cDNA, and by RNA mapping. The coding sequence of Cdc25A (Fig. 1) was determined as being expressed from 18,314 bp of genomic DNA comprising 15 exons.

WO 01/20034 PCT/USOO/24838 - 14 To make the foregoing determinations, genomic clones for DNA sequencing were isolated by hybridization screening of shot gun libraries prepared from a P1 vector containing the entire Cdc25A locus using oligonucleotides derived from a previously published cDNA sequence (Wickramasinghe, D., et al., (1995) Development 121, 2047 5 2056). Sequencing of these clones revealed that the transcribed portion of the Cdc25 gene is encoded within 18,314 bp of genomic DNA containing 15 exons, all of which contain coding regions. The exon structure of the Cdc25A gene is presented in Table 1. 10 Table 1. Exon structure and coding capacity of the mouse Cdc25A gene. Exon First Last Amino Genscan Nucleotide Nucleotide Acids Predicted 1 1 589 1-57 Yes 2 1589 1662 57-83 Yes 3 2707 2749 83-97 No 4 3662 3698 97-109 Sub-optimal 5 4265 4366 110-144 No 6 5904 6008 145-179 Yes 7 7929 8054 180-222 Yes 8 8559 8633 223-247 Sub-optimal 9 8782 8950 248-303 Yes 10 11688 11786 304-336 Yes 11 13315 13377 337-357 No 12 13473 13571 356-390 Yes 13 14152 14282 391-434 Yes 14 16187 16298 434-471 Yes 15 16521 18335 472-516 Yes Twelve of the fifteen exons where predictable using the Genscan gene 15 identification application (Burge, C., et al., (1997) JMol Biol 268(1):78-94) (http://bioweb.pasteur.fr/seqanal/interfaces/genscan-simple.html) (Table 1). No coding exons were predicted on the opposite strand. Intron size, exon size, and splice junction sequences are shown in Table 2.

WO 01/20034 PCT/USOO/24838 - 15 Table 2. Splicing junctions and intron structure of the mouse Cdc25A gene. Exon 3' Splice Donor 5' Splice Acceptor Exon Intron Size Size (bp) 1 TGGGCAGgtgagagacc 589 1009 2 ttggtttcagTGACTGT GATTCAGgtattgccat 64 2544 3 tttctcaaagGTTTCTG AAGAAAAgtatgtattc 43 912 4 ctcattacagCCTTGAA CCTACCTgtaagttctg 37 566 5 actttgatagCAGAAGC AGAAAATgtttgctgag 102 1537 6 tgttttgcagGAAGCAT TCGGATGgtacgtggtt 105 1920 7 tgtctttcagCTATCTT TATGAAGgtacagtctg 126 504 8 cttgctctagAATGATG AAACCTTgtaagttact 75 148 9 tgaccctaagGCCGATC CGCCCAGgttagttacc 169 2737 10 tttcttacagCTTCCAT CTCCAAGgtaactgcaa 99 1528 11 ttttctaaagGTTTATC AGAAATTgtaagtcagg 63 95 12 ctcttcatagATGGCAT CATCAAGgtttggattc 99 580 13 gtctccacagGGTGCCG CTCGAATgtgagtacca 131 1904 14 ttttcactagGTGCCGA GTGCCAGgtgagatgtc 112 222 15 gttcctttagTCTCACT 1815 Exon and intron sequences are shown in upper- and lowercase letters, respectively. The 5 splice acceptor and donor sequences are shown in boldface type. A canonical polyadenylation site AATAAA (Wickens, M., et al., (1984) Science 226(4678):1045-1051) was identified 17,893 bp down stream of the initiator methionine codon. The 3' most cluster of mouse ESTs present in the NCBI dbEST database was 10 determined to overlap this site, consistent with this being the 3' end of the gene. Previous reports have proposed a 3' end further upstream. This conclusion likely results from false priming of a poly A track present in the transcribed region. The 3' UTR contains two copies of the mRNA destabilizing motif ATTTA at positions 16,811 and 16,939 (Wilson, T., et al. (1988) Nature 336(6197):396-399). Theses motifs are conserved in the 15 corresponding sequence of the rat cDNA. The stability of Cdc25A mRNA in mouse cells was not determined however it is worth noting that in MCF7 cells, human Cdc25A mRNA has a half-life of approximately 2 hours.

WO 01/20034 PCTIUSOO/24838 - 16 Based on the genomic sequence and the sequence of the Cdc25A cDNA cloned from mouse kidney, the deduced amino acid sequence of murine Cdc25A was determined and is shown in Figure 1. This sequence differs at several positions form a previously published sequence that was based solely on cDNA sequence alone (Wickramasinghe, 5 D., et al., (1995) Development 121, 2047-2056). EXAMPLE 2 STRUCTURAL CHARACTERIZATION OF THE MURINE Cdc25A PROMOTER 10 In this example, a characterization of the murine Cdc25A promoter is presented. Base on the sequence determinations made above, the structure of the murine Cdc25A promoter could be determined. In particular, various key structural motifs of the Cdc25A promoter were identified. For example, the Cdc25A transcription initiation site was determined using SI nuclease protection analysis. Two genomic DNA probes were 15 used for this analysis, one labeled at the NcoI site coincident with the translation initiation site and one labeled at the NotI site 152 nucleotides upstream (Fig. 2A). As shown in Figure 2B, the NotI probe yields a protected fragment of approximately 260 nucleotides whereas the NcoI probe yields a product of approximately 420 nucleotides. These results place the transcription initiation about 420 nucleotides 5' of the translation 20 initiation site. This agrees roughly with the 5' end predicted based on the published sequence of the human Cdc25A cDNA (Galaktionov, K., et al., (1996) Nature 382, 511 517) although the exact sequence in the vicinity of the start site is not well conserved between mouse and human. This region does not contain a consensus TATA box but does contain the sequence AATTAA. This sequence is similar to degenerate TATA 25 elements shown to function, albeit very weakly, in yeast (Arndt et al., 1994, Mol. Cell Biol. 14:3719-3728). TATA independent transcription initiation can be directed by "initiator" (Inr) elements containing the consensus sequence PyPyAN(T/A)PyPy (Javahery et al., (1994) Mol Cell Biol (14(1):116-127). A perfect match to this consensus is found at position -420 relative to the translation initiation site. Inr sequence elements 30 are frequently associated with adjacent Spi binding sites (Faber et al., 1993, J. Biol. Chem. 268:9296-9301). Consistent with this, an SpI binding site is located at -40 relative to the Cdc25A transcription initiation site and consensus Inr element.

WO 01/20034 PCT/US00/24838 - 17 Immediately adjacent to the putative SpI binding site is a binding site for E2F that has been reported to mediate cell cycle arrest by TGF P in keratinocytes (Iavarone, A., et al., (1999) Mol Cell Biol 19:916-922). In addition, it has been observed that human Cdc25A reporter gene activity is reduced 75% during cell cycle arrest induced by 5 treatment with TGF P (laverone, A., et al. (1999) Mol Cell Biol 19:916-922). In addition to the identification of the foregoing motifs, a GRAIL prediction analysis (http://grail.lsd.ornl.gov/Grail-1.3/) was performed and it was determined that various CpG islands exist between nucleotides 852 and 1612, the region roughly encompassing the transcription initiation site through the end of exon one. 10 EXAMPLE 3 FUNCTIONAL CHARACTERIZATION OF THE Cdc25A PROMOTER In this example, a functional characterization of the murine Cdc25A promoter is presented. 15 To test for Cdc25A promoter function, a 1.3 Kb NotI Cdc25A genomic fragment was fused (+260/-1040 relative to the transcription initiation site) to the reporter gene luciferase. It has been observed that Cdc25A is expressed over a wide range of levels depending on tissue type. For example, expression is very high in the kidney but almost undetectable in the lung. In order to determine if this 1.3 Kb fragment could recapitulate 20 this expression pattern, transient transfection experiments were conducted. The Cdc25A luciferase construct was transfected into mammalian 293 kidney cells and NCI H460 lung cells in parallel with five other control constructs. These control constructs contained viral or house keeping gene promoters having little cell type specificity. As shown in Figure 3, the relative activity of the control constructs differs 25 very little between the two cell types. However, although Cdc25A driven luciferase activity was detected in both cell types, it was at least ten times higher in kidney cells than lung cells relative to the panel of control reporter constructs. These data suggest that cell type specific expression levels can be conferred by Cdc25A upstream sequences extending to approximately 1 Kb 5' of the transcription 30 initiation site.

WO 01/20034 PCT/USOO/24838 - 18 In addition to tissue specific regulation of the murine Cdc25A promoter, the autoregulation of the Cdc25A promoter was investigated. Accordingly, cells were cotransfected with the above-described mammalian luciferase reporter construct driven by the Cdc25A promoter and a plasmid encoding either a catalytically active Cdc25A 5 phosphatase or a catalytically inactive phosphatase. Transcription from the Cdc25A promoter was repressed by over-expression of catalytically active but not catalytically inactive Cdc25A phosphatase. Accordingly, it was concluded that the catalytically active Cdc25A phosphatase can repress the transcriptional activity of the Cdc25A promoter. This result may be 10 autoregulated by a mechanism of Cdc25A-mediated promoter suppression via Cdc25A phosphatase activity. This aspect of promoter suppression was further explored as a screening assay for modulators of Cdc25 phosphatase and these results are described in Example 4. 15 EXAMPLE 4 ASSAY FOR SCREENING MODULATORS OF CDC25A REGULATION OF CELLULAR AND VIRAL GENE TRANSCRIPTION In this example, an assay for measuring Cdc25-mediated gene regulation an identifying modulators thereof, is presented. 20 It has been observed that Cdc25A can affect the expression of genes containing binding sites for the transcriptional repressor CutI (Coqueret, 0., et al., (1998) EMBO Journal 17, 4680-4694). For example, Cdc25A can dephosphorylate Cutl thereby increasing its affinity for binding sites in promoters such as the p21/Waf gene. In addition, based on the functional characterization of the Cdc25A promoter in 25 Example 3, it was discovered that the Cdc25A phosphatase is capable of regulating the Cdc25A promoter. Accordingly, an investigation of Cdc25A-mediated gene regulation of a number of different eukaryotic promoters was conducted in addition to analysis of Cdc25A autoregulation. In particular, Cdc25A was assayed for the ability to regulate the promoter of an important cell cycle gene, i.e., p21, the phosphoglycerate kinase (pGK) 30 promoter, the promoter of a tissue restricted gene, i.e., p-globin, and a viral promoter derived from SV40.

WO 01/20034 PCT/USOO/24838 - 19 Accordingly, mammalian cells were co-transfected with one of several reporter constructs and a plasmid encoding either a catalytically active or catalytically inactive Cdc25 phosphatase. Following transfection, cells were harvested, and reporter gene activity as a function of luciferase activity was determined as described in the materials 5 and methods subsection above. As shown in Figure 4A, catalytically active Cdc25A was shown to repress p21 promoter expression in transient transfection experiments. Similarly, the Cdc25A promoter was inhibited by catalytically active Cdc25A but not catalytically inactive Cdc25A. Sequence analysis of the Cdc25A promoter revealed the presence of consensus 10 Cutl binding sites at -625, -551 and -482 (Andres, V., et al., (1994) Genes Dev 8(2):245 257). This indicated that Cdc25A expression could repress its own expression through a feed-back mechanism. The effects of Cdc25A phosphatase expression on the phosphoglycerate kinase (pGK) gene promoter and the p-globin promoter (-108) were also analyzed (Figure 4A). The results show that the catalytically active but not the 15 catalytically inactive form of Cdc25A was able to repress the pGK promoter but not the globin gene construct which does not contain Cutl binding sites. In order to determine if Cdc25A could affect the gene transcription of other promoters, a viral promoter derived from SV40 (simian virus 40) was tested. As shown in Fig 4 (lower panel), the catalytically active form of the Cdc25A phosphatase, but not 20 the catalytically inactive form, was able to repress transcription of the SV40 promoter and this was an extremely robust level of repression (42 fold repression). To verified that the 293 cells were transfected with equivalent amounts of constructs encoding Cdc25A, immunoblot analysis of Cdc25A polypeptide levels using polyclonal antisera was performed (Figure 4B). 25 Thus, these results show that Cdc25A expression can regulate gene expression including Cdc25 expression via the phosphatase activity of the Cdc25 polypeptide. This level of regulation is likely to be important in the regulation of cycling cells especially, e.g., in concert with other forms of regulation affecting, e.g., E2F activity (laverone, et al., (1999) Mol Cell Biol 19:916-922).

WO 01/20034 PCT/USOO/24838 - 20 These experiments further demonstrate the ability of Cdc25A to regulate not only different cellular promoters, such as its own promoter (but not all cellular promoters, see, e.g., the globin promoter), but also viral promoters, such as is exemplified using the SV40 promoter. 5 Accordingly, it will be appreciated that the assay has wide utility in screening modulators of Cdc25-mediated gene regulation. In particular, the viral promoter SV40 may be used because of the unambiguous signal that can be assayed and because an inhibitor of Cdc25A will rescue signal output, i.e., reporter gene expression. Because the amount of Cdc25A repression of this promoter is 42-fold, even weak or partial inhibitors 10 of Cdc25A activity can be readily assayed. Moreover, the assay provides a control that can accurately identify compounds that are false positives (e.g., compounds that rescue the signal but also increase the signal in the test reaction) or false negatives (e.g., compounds that produce no signal but also lower the control signal, e.g., cytotoxic compounds) and this insures that 15 inappropriate compounds are not further investigated and that candidate compounds or not erroneously dismissed. It will be further appreciated that any art recognized compound or library of compounds containing, e.g., a test compound that is protein based, carbohydrate based, lipid based, nucleic acid based, natural organic based, synthetically derived organic 20 based, or antibody based may be screened as a candidate compound that affects Cdc25 medated regulation of a promoter such as, e.g., the SV40 promoter. Accordingly, any of a number of art recognized high throughput assay techniques may be used in conducting the assay. 25 EXAMPLE 5 CHARACTERIZATION OF CELL CYCLE REGULATION OF CDC25A EXPRESSION In this example, the cell cycle regulation of Cdc25A is described. To determine if murine Cdc25A levels changed during the cell cycle, Cdc25A 30 RNA levels were measured during the cell cycle of murine cells synchronized with a double thymidine block (Pagano, M. (ed.) Cell Cycle - Materials and Methods. New York: Springer-Verlag, 1995). Figure 5 shows that, relative to the level of Cdc25A WO 01/20034 PCT/US00/24838 -21 mRNA in cells arrested at Gl/S (0 hours), Cdc25A mRNA levels peak at 9 hours after release corresponding roughly to the time when most cells are in G2 or M phase. It was also observed that mRNA levels begin to drop off as cells continue to finish the first cell cycle after release. These results are consistent with observations made of human 5 Cdc25A protein levels using HeLa cells and NCI H460 cells. In addition, it has been observed that Cdc25A mRNA levels are cell cycle regulated in rat NRK cells (Jinno, S., et al., (1994) EMBO Journal 13, 1549-1556). Thus, consistent with rat and human cells, murine Cdc25A levels appear to be modulated by factors affecting cell cycle progression. Moreover, these findings indicate 10 that the mouse Cdc25A is expressed over a wide range, in a cell type specific manner, and its expression can be affected by check-point arrest. To explore the role of Cdc25A in vivo, transgenic animals were prepared as described in Example 7. EXAMPLE 6 15 NOVEL VECTORS FOR CDC25A GENE TARGETING In this example, the construction and recombination frequencies of gene targeting vectors constructed using the above-mentioned sequence information are described. The availability of genomic sequence from the Cdc25A locus facilitated the construction of novel gene targeting vectors for disrupting the Cdc25A locus. First, a 20 conventional targeting vector was constructed and designed to remove portions of exon 1 and exon 2 resulting in disruption of the coding sequence (Figure 6). Additionally, a vector was constructed containing a cassette (tNT) designed to simultaneously insert the coding sequences of the tetracycline trans-activator (Gossen, M., et al., (1993) Trends Biochem Sci 18(12):471-475), under control of the Cdc25A promoter and place the 25 adjacent coding sequences of the Cdc25A gene under the control of the tetracycline response element tet-o-7 (Gossen, M., et al. (1993) Trends Biochem Sci 18(12):471-475) (Figure 6). The second construct was expected to conditionally express Cdc25A based on the presence or absence of tetracycline. The embryonic cell line EL1 was electroporated with each of these constructs and placed under selection for G418 30 resistance (negative selection was not used). Surprisingly, the construct containing the tNT insertion frequency was high (over 25% (14/54)) (Figure 6). Although recombination between one arm of the conventional targeting vector and the Cdc25A WO 01/20034 PCT/USOO/24838 - 22 locus was detected, no homologous recombinants were identified among 210 G418 resistant clones (Figure 6). These results may indicate that sequences within exons 1, 2, and 3 may influence homologous recombination frequency or are necessary for genetic stability of the locus. 5 Accordingly, it was determined that relatively subtle differences in the design of Cdc25 targeting vectors can have a significant impact on recombination frequencies. EXAMPLE 7 PREPARATION AND ANALYSIS OF MICE LACKING A CDC25 10 PHOSPHATASE In this example, the preparation and analysis of transgenic mice lacking a Cdc25 polypeptide, is described. Cyclin Dependent Kinase (CDK) activity controls cell division in eukaryotes and is positively regulated by CDC25, a family of dual specificity phosphatases. In mice, 15 three Cdc25 genes, Cdc25A, B and C, have been identified and are expressed in an overlapping yet distinct manner during development (Sadhu, K., et al. (1990) PNAS USA 87:5139-5143; Kakizuka, A., et al. (1992) Genes and Development 6:578-590; Wickramasinghe, D., et al. (1995) Development 121:2047-2056; Wu, S., et al. (1995) Dev Biol 170:195-206). To examine the specific roles and potential redundancy of these 20 genes during mouse development, Cdc25A deficient mice were generated. A lethal phenotype is observed in Cdc25A deficient embryos in contrast to that of Cdc25B mutants that remain viable. These results indicate that Cdc25A is essential and is not redundant for early mouse development, in contrast to that of Cdc25B, unequivocally distinguishing the unique role played by these individual family members. 25 Targeting Strategy The Cdc25A locus was targeted by site directed mutagenesis in embryonic stem cells. Exon 1 was disrupted by insertion of a PGK-Neomycinr (PGK-NEOr) cassette containing three polyadenylation signals and translation termination codons in all three 30 reading frames. The targeting vector, which contained 1.3kb and 3.2kb of 5' and 3' homology regions respectively (Figure 7), was electroporated into Embryonic Stem (ES) cells and selected for resistance to 300ptg/ml G418. No negative selection was WO 01/20034 PCT/USOO/24838 - 23 performed. Homologous recombination in these clones was identified by Southern blot analysis and 3 independent cell lines were expanded. These clones were injected into blastocysts and transferred to recipient mothers. Chimeric mice were identified and crossed with C57BL6 mice. Heterozygous offspring obtained from these crosses were 5 healthy and fertile and have remained so through the 9-month duration of this analysis. Multiple litters of heterozygous intercross spring derived from the three different cell lines were genotyped. No homozygous mutant mice were identified although heterozygous and wildtype offspring were represented (Table 3). These observations suggested that the homozygous mutant phenotype was associated with embryonic 10 lethality. Table 3. Genotype analysis of Cdc25A heterozygous intercross offspring. Wild type Heterozygous Homozygous Total #2 5 11 0 16 #9 10 22 0 32 #36 32 59 0 91 15 Three independent lines (#2, # 9 # 36) of Cdc25A heterozygous intercrossed offspring were genotyped by Southern blot analysis using the 3'probe P2 described in Figure 7. In no case were homozygous null offspring identified. Phenotypic Analysis 20 In order to identify the developmental stage at which the embryonic lethality occurs, various stages of embryos were dissected from heterozygous intercrosses ranging from E6.5 to E12.5. Examination of E6.5 embryos indicated that all embryos developed to the egg cylinder stage and appeared normal (n = 57). However, at E7.5 disorganized embryos undergoing resorption were observed consistently in 25% of the 25 embryos analyzed (n = 62). Embryonic death was observed in E8.5 and later stages of development at a similar ratio (n = 100). Histological examination of embryos from heterozygous intercrosses at E6.5 and E7.5 confirmed these observations (Figure 2). In addition, control crosses with wildtype and heterozygous mice do not display embryonic lethality at E7.5 in contrast to the heterozygous intercross embryos.

WO 01/20034 PCT/USOO/24838 - 24 These observations indicate that wildtype and heterozygous embryos do not contribute to the embryonic lethal phenotype. Wildtype embryos at E6.5 have developed to the egg cylinder stage (M. H. Kaufman (1995) The Atlas ofMouse Development, Acad. Press. p1-41). All 5 heterozygous intercross embryos develop to this stage and are indistinguishable from each other (Figure 8A-J). At E7.5 normal embryos develop to the late primitive streak stage. The presence of ectoderm, mesoderm and endoderm layers, in addition to the chorion, allantois, amnion and neural plate easily distinguish embryos at this stage (Figure 8K-Q). However, in contrast, we observed approximately 25% of the embryos 10 were disorganized and did not display distinct embryonic layers or neural plate formation that resulted in early embryonic lethality. In Situ Hybridization Analysis To examine the embryonic lethality in greater detail, in-situ hybridization 15 analysis was carried out on E7.5 embryos (as described in J. Coligan et al., (1993) Cur. Protocol Immunol (Wiley ) Unit 12.8.1 - 21). Cdc25A RNA was detected ubiquitously in all embryos that appeared normal and had developed to the late primitive streak stage (Figure 2C, C'). Overall Cdc25A expression was observed in the endoderm, mesoderm, and ectoderm layers, the ectoplacental cone, extra-embryonic layers, and in the allantois, 20 amnion and neural plate. An adjacent section of the same embryo hybridized to a cda25b probe revealed lower overall expression but specific localization of Cdc25B RNA to a subset of extra-embryonic mesodermal cells (Figure 8D, D'). In contrast, the disorganized embryos do not express Cdc25A (Figure 9E, E') in the ubiquitous expression pattern observed in normal sibling embryos. However, Cdc25B RNA was 25 detected in an adjacent section of the same embryo (Figure 9F, F'). Although the embryonic tissue is disorganized the specific expression of Cdc25B is easily distinguishable, while Cdc25A expression cannot be detected above background levels. These observations reveal that Cdc25A expression (wildtype or heterozygous) is correlated with development of the embryo while the lack of Cdc25A expression is 30 directly correlated with early embryonic lethality. Therefore, Cdc25A is essential and is not redundant for embryonic development.

WO 01/20034 PCT/USOO/24838 - 25 Since phenotypes have been identified in mice heterozygous for other cell cycle genes, we examined cell cycle profiles and DNA damage response in mice heterozygous for Cdc25A (A. Clarke et al. (1992) Nature 359: 328-330; L. Donehower et al., (1992) Nature 356: 215-221; T. Jacks et al.(1992) Nature 359: 295-300). Cell cycle profiles, 5 by FACS analysis, of synchronized or DNA damaged heterozygous Mouse Embryo Fibroblasts (MEFs) were indistinguishable from their wildtype counterparts. Furthermore, Northern and immunoblot analysis of embryonic RNA and protein respectively, reveals similar levels of Cdc25A in wildtype and heterozygous embryos. cDNA expression arrays also indicate similar expression 10 patterns in wildtype and heterozygous embryos and corroborate our previous Northern analysis data. These results unequivocally demonstrate that Cdc25A is essential for early embryonic development. At E7.5 overall developmental arrest is observed in mutant embryos suggesting that Cdc25A plays a critical role at this time of rapid proliferation. 15 These results further distinguish the role of Cdc25A from that of Cdc25B. Since Cdc25B has been implicated in a role at G1/S, compensation for the lack of Cdc25A function by Cdc25B at G,/S would be predicted. However, the Cdc25A mutant embryos are not compensated for by Cdc25B, although it could be argued that the low level of Cdc25B expressed in these embryos is insufficient for compensation. Furthermore, the 20 Cdc25A mutant phenotype is in sharp contrast to that of Cdc25B. Cdc25B is not essential or is redundant in embryonic development. Cdc25B -/- mice develop into adult animals and do not display a mitotic phenotype. These observations suggest that Cdc25A plays a central role in early embryogenesis co-incident with tissue proliferation for subsequent development and 25 differentiation. Zygotic expression of Cdc25A occurs most likely at the blastocyst stage of development (D. Wickramasinghe et al., (1995) Development 121:2047-2056). Therefore, Cdc25A homozygous mutants probably survive preimplantation development, due to maternally provided Cdc25A. Alternatively, Cdc25A is not essential for these early embryonic divisions. Maternal support of early development 30 has been documented extensively and survival of pre-implantation embryos is not unique in mouse knockout analysis (Telford, N., et al., (1990) Mol Reprod Dev 26:90 100). For example, cyclin A2 -/- embryos express the protein in blastocysts. The cyclin WO 01/20034 PCT/US00/24838 - 26 A2 is most likely derived from maternal stores and supports embryonic development up to E6.5 (Murphy, M. et al. (1997) Nature Genetics 15:83-86). In addition, knockout embryos of genes critical for cell division and DNA damage repair, such as BRCA1, BRCA2 and RAD51 display early embryonic lethal phenotypes ranging from E6.5-8.5 5 (Gowen, L., et al. (1996) Nature Genetics 12:191-194). The results presented here describe the pivotal role Cdc25A plays during mouse development that is temporally coincident with rapid proliferation. Since Cdc25A has been implicated in numerous human cancers, these mutants extend a framework to examine genetic interactions with other cell cycle regulators and tumor suppressors in 10 creating a malignant state. In particular, these cells and resultant animals provide valuable tools for assaying Cdc25-mediated gene regulation. Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than 15 routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. What is claimed: 20

Claims (21)

1. A method for identifying an modulator of Cdc25 activity comprising, providing a cell having a recombinant Cdc25 phosphatase gene wherein the 5 expression of said gene alters the transcription of a selected gene; contacting the test cell with a compound under conditions wherein said recombinant Cdc25 phosphatase gene is expressed and alters the transcription of a selected gene as an indication of said compound being a modulator of Cdc25-mediated transcription. 10

2. The method of claim 1, wherein the method further comprises providing a control test cell having a recombinant catalytically inactive Cdc25 phosphatase gene, wherein the expression of said phosphatase does not alter the transcription of a selected gene; 15 contacting the control test cell with a compound under conditions wherein said recombinant catalytically inactive Cdc25 phosphatase gene is expressed; and comparing the amount of transcription of the selected gene in the presence of the compound to the amount of transcription occurring in the absence of the compound wherein a statistically significant change in the amount of transcription of the selected 20 gene in the presence of the compound is indicative of said compound being a modulator independent of Cdc25-mediated transcription.

3. The method of claim 1, wherein said selected gene comprises a eukaryotic promoter element. 25

4. The method of claim 1, wherein said selected gene is selected from the group consisting of p21/WAF, pGK, and Cdc25.

5. The method of claim 1, wherein the selected gene comprises a reporter gene. 30 WO 01/20034 PCT/USOO/24838 - 28

6. The method of claim 5, wherein the reporter gene is luciferase.

7. The method of claim 5, wherein the reporter gene is controlled by a cellular promoter. 5

8. The method of claim 6, wherein said cellular promoter is selected from the group consisting of p2l/WAF, pGK, and Cdc25.

9. The method of claim 7, wherein said cellular promoter is the p21/WAF 10 promoter.

10. The method of claim 7, wherein said cellular promoter is the pGK promoter.

11. The method of claim 7, wherein said cellular promoter is a Cdc25 promoter. 15

12. The method of claim 5, wherein the reporter gene is controlled by a viral promoter

13. The method of claim 12, wherein the viral promoter is the SV40 promoter. 20

14. The method of claim 1, wherein the recombinant Cdc25 phosphatase gene encodes a mammalian Cdc25 phosphatase.

15. The method of claim 14, wherein the mammalian Cdc25 phosphatase is a human 25 Cdc25 phosphatase.

16. The method of claim 15, wherein the human Cdc25 phosphatase is selected from the group consisting of Cdc25A, Cdc25B, and Cdc25C. 30

17. The method of claim 1, wherein the test cell is a mammalian cell.

18. The method of claim 17, wherein the test cell is a human cell. WO 01/20034 PCT/USOO/24838 - 29

19. The method of claim 6, wherein said transcription is determined by measuring luciferase activity.

20. The method of claim 1, wherein said change is an increase indicating said 5 compound is an inhibitor of Cdc25 activity.

21. The method of claim 1, wherein said change is a decrease indicating said compound is an activator of Cdc25 activity. 10