WO2002068386A2

WO2002068386A2 - Tools and methods for identifying ppar$g(d)-specific agonists and antagonists

Info

Publication number: WO2002068386A2
Application number: PCT/US2002/003408
Authority: WO
Inventors: Ben Ho Park; Kenneth W. Kinzler; Bert Vogelstein
Original assignee: The Johns Hopkins University
Priority date: 2001-02-27
Filing date: 2002-02-27
Publication date: 2002-09-06
Also published as: WO2002068386A3; AU2002247081A1

Abstract

Cells which are PPARδ gene-defective can be used together with isogenic PPARδ gene wild-type cells to identify PPARδ-specific agonists and antagonists. PPARδ-specific agonists can be used, inter alia, to treat inflammation, to improve fecundity, or to ameliorate toxic effects of non-steroidal anti-inflammatory drugs. PPARδ-specific antagonists can be used, inter alia, to treat cancer and other disorders characterized by increased cell proliferation. Lipid homeostatis also can be modulated using PPARδ-specific agonists and antagonists.

Description

TOOLS AND METHODS FOR IDENTIFYING PPARδ -SPECIFIC AGONISTS AND ANTAGONISTS

This invention was made using grant funds from the U.S. National Institutes of Health (CA57345 and CA62924). Therefore the government retains some rights in the present invention.

TECHNICAL FIELD OF THE INVENTION

The invention relates to the area of screening assays for therapeutic agents. More particularly, the invention relates to the area of screening assays for PPARδ- specific agonists and antagonists.

BACKGROUND OF THE INVENTION

The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors originally identified by their ability to mediate the transcriptional effects of peroxisome proliferators (reviewed in 1). Subsequently, PPARs have been implicated in many normal and disease related processes including lipid metabolism, inflammation, embryo implantation, diabetes, and cancer (reviewed in 2). Similar to other nuclear hormone receptors, PPARs heterodimerize with another nuclear hormone receptor, RXR, and exert their effects via regulation of gene transcription upon binding of ligand. To date, three isotypes have been identified: α, γ, and δ (the latter also known as PPARβ and NUC-1). Although all three PPAR isotypes modulate lipid metabolism, isotype-specific functions have also been described. For example, on the basis of experiments with specific agonists and knock out mice, PPARγ has been shown to be involved with insulin resistance (3). PPARγ also has been implicated in a variety of neoplastic processes, including colorectal cancer, although its role as a promoter or suppressor of tumor growth has remained controversial due to conflicting results in mouse versus human systems (4-6).

Recently PPARδ was identified as a potential downstream target of the APC/β- catenin/TCF-4 tumor suppressor pathway (7). In normal colorectal tissue, APC binds to β- catenin and inhibits its ability to form a bipartite transcription complex with TCF-4 (8-10). In the majority of colorectal cancers, APC is inactivated by truncating mutations, thus giving rise to elevated levels of β-catenin/TCF-4 mediated transcriptional activity. Mutations of β- catenin that render it resistant to the inhibitory effects of APC often occur in cancers with intact APC genes, strongly suggesting that inhibition of β-catenin/TCF-4 mediated transcription is critical to APC's tumor suppressive effects (10-12). Several targets of the β-catenin/TCF-4 transcription complex have been identified, including c-MYC (13) and Cyclin Dl (14), which have obvious growth promoting properties. PPARδ was identified as another potential target of this pathway after it was shown to be down regulated upon restoration of APC expression in a human colorectal cancer cell line (7). Because the PPARδ promoter contains TCF-4 binding sites and PPARδ promoter reporters are repressed by APC as well as stimulated by mutant β-catenin, this PPARδ down regulation appears to be direct. Consistent with PPARδ' s role as an APC/β-catenin target, PPARδ mRNA has been found to be consistently over expressed in many colorectal cancers (7, 15). Finally, the ability of nonsteroidal anti-inflammatory drugs (NSATDs) to bind PPARδ (16) and potentially inhibit its function (7) suggested that inhibition of PPARδ might contribute to the chemopreventative effects of NSAIDs such as sulindac. Tools and methods of identifying PPARδ-specific antagonists and agonists would, therefore, be useful for identifying molecules which can be administered to provide therapeutic effects.

SUMMARY OF THE INVENTION It is an object of the present invention to provide tools and methods of screening for

PPARδ-specific antagonists and agonists. This and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is a homozygous PPARδ gene-defective cell line. Another embodiment of the invention is a pair of isogenic cell lines. Cells of a first cell line are homozygous PPARδ gene-defective, and cells of a second cell line are homozygous PPARδ gene wild-type.

Yet another embodiment of the invention is a method of identifying a test compound as a PPARδ-specific antagonist or agonist. A first and a second isogenic cell are contacted with a test compound. The first cell is a homozygous PPARδ gene-defective cell, and the second cell is a homozygous PPARδ gene wild-type cell. Rate of division of the first and second cells is assessed. A test compound that reduces the rate of division of the second cell relative to the first cell is identified as a PPARδ-specific antagonist. A test compound that enhances the rate of division of the second cell relative to the first cell is identified as a PPARδ-specific agonist.

Even another embodiment of the invention is a method of identifying a test compound as a PPARδ-specific antagonist or agonist. A test compound is administered to a first and second experimental animal. The first and second animals are provided with xenografts. The first animal is provided with a xenograft comprising homozygous PPARδ gene-defective tumor cells, and the second animal is provided with a xenograft comprising PPARδ gene wild-type tumor cells. The PPARδ gene-defective and the PPARδ gene wild-type cells are isogenic. Tumorigenicity of the PPARδ gene-defective and the PPARδ gene wild-type cells is assessed. A test compound that reduces the tumorigenicity of the PPARδ gene wild-type cells relative to the PPARδ gene-defective cells is identified as a PPARδ-specific antagonist. A test compound that enhances the tumorigenicity of the PPARδ gene wild-type cells relative to the PPARδ gene-defective cells is identified as a PPARδ-specific agonist. The invention thus provides tools and methods for identifying PPARδ -specific antagonists and agonists. Such molecules provide potential therapeutic agents for the treatment of disorders such as cancer and inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Strategy for generating PPARδ null somatic cell lines. FIG. 1A. Constructs used to mediate recombination at the PPARδ locus. PKOl contained a 1 kb 5' homology arm and a 4 kb 3' homology arm and was used to generate an 11 kb deletion encompassing the entire DNA binding domain (exons 4, 5, and 6). PKO2 contained a 1.8 kb 5' homology arm and a 4 kb 3' homology arm and was used to delete exon 4. The endogenous PPARδ locus, the locus after targeting with PKOl, and the targeted locus after Cre mediated excision are illustrated. Lx and Hyg-TK designate LoxP sequences and the hygromycin/thymidine kinase gene, respectively. The thymidine kinase gene was not used for negative selection in the protocols actually applied in this study. Apa I sites and the probe sequence used for Southern blotting are indicated. FIG. IB. Schematic of the PPARδ rescue vector. An HAHA-tagged cDNA of PPARδ was cloned into a construct containing an IRES-geneticin resistance gene cassette flanked by LoxP sequences as described in Example 1. FIG. 2. Southern Blot Analysis of PPARδ engineered cell lines. "HCT116," the parental cells; "PPARδ +/-," HCT116 cells after successful recombination with PKOl;

"Cre'd PPARδ +/-," PPAR +/- cells after Cre mediated LoxP excision of the Hyg-TK gene;

"PPARδ -/-," PPARδ +/- cells after successful recombination of the remaining PPARδ locus with PKOl.

FIG. 3. PPARδ is not required for sulindac mediated apoptosis. HCT116, PPARδ +/- , and PPARδ -/- clones were treated for 72 hours with 0, 80, 100, and 125 μM of sulindac sulfide. Surviving colonies were assessed by crystal violet staining. Treatments were performed in triplicate, and representative wells are shown. FIG. 4. PPARδ is required for efficient tumorigenicity. HCT116, PPARδ+/-, and

PPARδ-/- cells were injected as xenografts in nude mice and sacrificed after 34 days as described in Example 1. FIG. 4A. Representative mice at day 34. FIG. 4B. Growth curves of HCT116, PPARδ+/-, and PPARδ-/- xenografts. Each data point for HCT116 represents the average tumor size of 6 mice, while each data point for the PPARδ+/- and PPARδ-/- cell lines represents the average tumor size of five mice. Error bars represent the standard error of the mean. Tumors that did not grow were assigned a volume of zero.

FIG. 5. In vitro growth characteristics of PPARδ modified cells. FIG. 5A. In vitro growth assay comparing growth kinetics of parental HCT116, PPARδ +/-, and PPARδ -/- clones. Each data point represents the average of three wells, with the bars representing the standard deviation from the mean. FIG. 5B. Colony formation assay demonstrating no difference in colony number between parental HCT116, PPARδ +/-, and PPARδ -/- clones. Colony numbers were roughly equivalent among all four cell lines although the parental HCT116 colony sizes were slightly larger, consistent with the growth curves in FIG. 5A.

FIG. 6. RXR and PPARδ consensus binding sites. FIG. 6A. RXR consensus binding site. PCR products of a randomized oligonucleotide template that bound a GST fusion protein containing the DNA binding domain of RXR were selected, cloned, and sequenced. The sequences of twenty-eight clones are shown (SEQ ID NOS:22-49), manually aligned to derive the consensus binding sequence (SEQ ID NO: 50) indicated at the bottom. FIG. 6B. PPARδ consensus binding site. PCR products of a randomized oligonucleotide template that bound a GST fusion protein containing the DNA binding domain of PPARδ were selected, cloned, and sequenced. The sequences of twenty clones are shown (SEQ ID NOS: 1-20), manually aligned to derive the consensus binding sequence indicated at the bottom (SEQ ID NO:21). FIG. 6C. Binding Specificity of PPAR , PPARδ and PPARγ. Oligonucleotides containing the indicated binding elements (DRE or ACO) were P-labeled and incubated with GST fusion proteins containing either the PPARα, PPARδ, PPARγ, RXR, or no DNA binding domain (-). DNA binding was assessed by GEMSA, where "Probe" indicates the unbound probe and "Shifted" indicates bound probe. FIG. 6D. DRE confers PPARδ responsiveness. The 293 human cell line was transfected with the indicated (DRE or ACO) luciferase reporters (0.3 μg), with a β-galactosidase expression vector (0.2 μg pCMNβ), and with 1.0 μg of either a vector control (Vector), PPARδ, or PPARγ expression vectors. Luciferase activity is reported relative to the vector control after normalizing for transfection efficiency through β-galactosidase activity. Bars represent the means of three independent replicates with the error bars representing the unbiased standard deviations.

DETAILED DESCRIPTION It is a discovery of the present invention that isogenic PPARδ gene-defective and

PPARδ gene wild-type cells are particularly useful for identifying PPARδ-specific agonists and antagonists. The development of agents that specifically target PPARδ can lead to more efficacious and less toxic therapeutic agents. Treatment of any condition currently treated with NSAIDs or prostacyclins can be achieved using PPARδ-specific antagonists or agonists, respectively. PPARδ-specific antagonists are potential therapeutic agents for use in treating cancer, in preventing polyp development, and in treating other conditions in which decreased cellular proliferation is desired, such as hyperplastic or dysplastic conditions. PPARδ- specific agonists are potential therapeutic agents for use in treating inflammation, improving fecundity, ameliorating toxic effects of non-steroidal anti-inflammatory drugs (NSAIDs), in treating conditions in which cells are dying prematurely (e.g., Alzheimer's Disease, AIDS, muscular dystrophy, amyotrophic lateral sclerosis, or other muscle wasting diseases, autoimmune diseases, heart attack, stroke, ischemic heart disease, lddney failure, septic shock, or a disease in which the cell is infected with a pathogen, such as a virus, bacterium, fungus, mycoplasm, or protozoan). Modulation of lipid homeostasis (e.g., control of cholesterol levels) can be achieved either with PPARδ - specific agonists or antagonists. _ __

PPARδ-specific antagonists would be particularly useful for treating cancer. PPARδ is over expressed in both human and rodent colorectal tumors (7, 15). The generation of PPARδ gene defective cells, such as the PPARδ -/- colon cancer cell line described herein, permit the identification of PPARδ-specific antagonists useful for treating cancer. PPARδ is an especially good target for pharmaceutical development because it is a receptor for ligands that can be modified to form antagonists or agonists. Moreover, it is a member of a family of genes that are eminently "drug targetable" and that form the targets for a large number of current drugs used to treat a variety of diseases. Specific pharmacologic inhibitors can be developed against PPARδ, which will inhibit the in vivo growth of tumors in ways similar to that observed after genetic disruption of this gene, described in the specific examples.

PPARδ gene-defective and PPARδ wild-type cell lines

A "PPARδ gene-defective cell" lacks one or two wild-type PPARδ gene alleles. Lack of two PPARδ wild-type alleles may result, for example, in diminished expression of a PPARδ gene or expression of a less functional PPARδ protein. In a preferred embodiment, a PPARδ gene-defective cell is homozygous, i.e., it lacks both wild-type PPARδ gene alleles and is PPARδ null.

Any means known in the art to generate a cell line which is defective in a PPARδ gene can be used to obtain PPARδ gene-defective cells. For example, promoterless homologous recombination can be used to generate an isogenic PPARδ negative cell line (Waldman et al., Cancer Res. 55, 5187-90, 1995). Any type of mammalian cell that can be maintained in culture or in an animal and can be transfected can be used to generate a PPARδ gene-defective cell. These cells include, but are not limited to, primary cells, such as fibroblasts, myoblasts, leukocytes, hepatocytes, endothelial cells, and dendritic cells, as well as cell lines (e.g., NCI-BL2126, Hs 578Bst, HCC1954 BL, Hs 574.Sk, Hs888Lu, which are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209). Preferred cells include tumor cells, preferably human tumor, more preferably human colon tumor cells. Appropriate cells are, for example, colorectal cancer cells, present either in situ in a mammalian body or in vitro in a tissue culture preparation. Tumor cells can be isolated from patients and placed in tissue culture. Alternatively, established tumor cell lines, such as HT29, SW480, HCT116, DLD1, MCF-7, HL-60, HeLa cell S3, K562, MOLT-4, Burldtt's lymphoma Raji, A549, G361, M12, M24, M101, SK-MEL, U-87 MG, U-118 MG, CCF-STTGl, or SW1088 can be used.

For purposes of the present invention, a cell with two wild-type alleles of a PPARδ gene is a "PPARδ gene wild-type cell." Preferably the PPARδ gene-defective cell used in methods of the invention is the same type of cell (i.e., originates from the same type of organ) as the PPARδ gene wild-type cell. More preferably, the two cells are isogenic. A pair of PPARδ gene-defective and PPARδ gene wild-type cell lines can be in a single, divided container, including, without limitation, a cell culture dish or flask, a liquid nitrogen container, a freezer box, a freezer, a refrigerator, a tissue culture hood, or an analytical device.

Cell lines comprising reporter constructs

The PPARδ gene-defective and PPARδ gene wild-type cells described above can host a report construct. Reporter constructs can be used to identify test compounds which act as

PPARδ-specific agonists or antagonists. Reporter constructs contain a PPARδ binding element. If desired, the reporter construct can include one or more RXR binding elements upstream of the minimal promoter.

The nucleotide sequence of the PPARδ binding element can be selected, for example, from any of the nucleotide sequences shown in FIG. 6B (SEQ ID NOS: 1-21), including the consensus nucleotide sequence CGCTCAC (nucleotides 3-9 of SEQ ID NO: 21) (see U.S.

Serial No. 09/638,623). PPARδ binding elements with other nucleotide sequences which bind PPARδ protein can also be used in reporter constructs. Such binding elements can be identified, for example, by carrying out assays which can detect PPARδ protein-DNA binding, such as DNA footprinting, electrophoretic mobility shift assays, or immunoprecipitation of PPARδ-DNA complexes using antibodies specific for PPARδ. Such methods are well known in the art.

The nucleotide sequence of the RXR binding element can be selected from any of the nucleotide sequences shown in FIG. 6A (SEQ ID NOS:22-50), including the consensus sequence GGTCA (nucleotides 3-7 of SEQ ID NO: 50). Other RXR binding elements which bind RXR can be identified as described for PPARδ binding elements, above. The PPARδ and RXR binding elements can be located directly adjacent to each other in the reporter construct, as shown in SEQ ID NO:71, or can be separated by any number of nucleotides which still permits functional binding of a PPARδ/RXR heterodimer, such as at least 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. If desired, the isolated reporter construct can comprise 1, 2, 3, 4, or more copies of the PPARδ binding element. Multiple copies of the RXR binding element can also be included.

Reporter constructs comprising PPARδ and RXR binding elements can be prepared using standard recombinant DNA techniques. Nucleic acid constructs can be linear or circular molecules, with or without replication sequences. Nucleic acid constructs contain 1, 2, 3, or 4 or more copies of the PPARδ binding element. A nucleic acid construct typically contains a reporter gene which encodes an assayable product, such as β-galactosidase, luciferase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), or chloramphenicol acetyltransferase (CAT). Many such reporter genes are known in the art.

The reporter gene can be under the control of a minimal promoter, such that in the absence of PPARδ the reporter gene is not expressed or is expressed only at low levels. Such reporter gene constructs can be used, for example, in methods for pre-screening test compounds to identify PPARδ-specific agonists and antagonists. In these reporter gene constructs, the minimal promoter is upstream from the reporter gene, and at least one copy of the PPARδ binding element is upstream from the minimal promoter. Optionally, 2, 3, 4, or more copies of the PPARδ binding element can be present. Suitable minimal promoters include, for example, the minimal CMV promoter (Boshart et al., 1985) and the promoters for TK (Nordeen, 1988), IL-2, and MMTV.

Methods of identifying PPARδ-specific agonists and antagonists

Various methods of identifying test compounds as PPARδ-specific agonists or antagonists can be performed. Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is typically used for polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

It is of particular interest to use as test compounds NSAIDs, such as sulindac, indomethacin, and other COX inhibitors (for other NSAIDs which can be tested, see Goodman & Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 9th ed, McGraw Hill, and Cada et al., FACTS AND COMPARISONS, J.B. Lippincott, 1999).

Methods of the invention can be carried out in vitro or in vivo. In one embodiment, isogenic homozygous PPARδ gene-defective cells and PPARδ gene wild-type cells are contacted with a test compound. Rate of division of the cells is assessed. A test compound that reduces the rate of division of the second cell relative to the first cell is identified as a PPARδ-specific antagonist, whereas a test compound that enhances the rate of division of the second cell relative to the first cell is identified as a PPARδ-specific agonist. Cell division can be assessed by any means known in the art including, but not limited to, manual or automated counting of cells in a standardized microscopic field, exposing the cells to tritiated thymidine and counting thymidine-labeled cells after carrying out autoradiography, or measuring DNA content.

The isogenic PPARδ gene-defective cells and PPARδ gene wild-type cells can contain a reporter construct, as described above. PPARδ gene-defective or PPARδ gene wild-type cells can be either stably or transiently transfected. Introduction of reporter constructs can be carried out in culture or in vivo. Methods of transfecting nucleic acid constructs into cells are well known and include, without limitation, transfection with naked or encapsulated nucleic acids, cellular ^"fusion, protoplast fusion, viral infection, and electroporation. After contacting the cells with a test compound, expression of the reporter gene is determined. A test compound that decreases the amount of expression of the reporter gene in the wild-type cells relative to the PPARδ gene-defective cells is identified as a PPARδ-specific antagonist, whereas a test compound which increases the amount of expression of the reporter gene in the wild-type cells relative to the PPARδ gene-defective cells is identified as a PPARδ-specific agonist.

Expression of the reporter gene can be determined by any method suitable for detecting the assayable product of the particular reporter gene used, including biochemical, immunological, or visual detection methods. Expression of the reporter gene can also be determined by detecting its mRNA, for example using Northern or dot blots or in situ hybridization. Expression of the reporter gene can be determined qualitatively or quantitatively, for example by reference to a standard curve. Preferably, the test compound decreases or increases expression of the reporter gene by at least 25, 50, 75, 85, 90, 95, 97, or 98 percent.

A test compound is administered to experimental animals, such as rats, mice, guinea pigs, or rabbits. The animals bear xenografts comprising either homozygous PPARδ gene- defective tumor cells or PPARδ gene wild-type tumor cells. The PPARδ gene-defective and the PPARδ gene wild-type cells are isogenic, so that any effects observed can be attributed to the presence or absence of the wild-type PPARδ gene. Alternatively, the test compound can be administered to the animals before the animals receive the xenografts, so that a preventative effect of the test compound can be determined.

Tumorigenicity of the tumor cells in the xenografts can be assessed by measuring, for example, number of tumors formed by the xenografts, rate of tumor formation by the xenografts, or size of tumors formed by the xenografts. A test compound that reduces the tumorigenicity of the PPARδ gene wild-type cells relative to the PPARδ gene-defective cells is identified as a PPARδ-specific antagonist. A test compound that enhances the tumorigenicity of the PPARδ gene wild-type cells relative to the PPARδ gene-defective cells is identified as a PPARδ-specific agonist. ' Using the isogenic cell pairs of the invention, the effect of test compounds on other down-stream sequelae of PPARδ can be assessed. Such sequelae include, but are not limited to, alterations in lipid profiles, myelination of the corpus callosum, lipid metabolism, epidermal cell proliferation, adipocyte differentiation, and uterine implantation. Peters et ah, Mol. Cell Biol. 20, 5119-28, 2000; Bastie et al, J. Biol. Chem.274, 21920-25, 1999; Xing et al., Biochem. Biophys. Res. Commun. 217, 1015-25, 1995; Matsuura et al, Mol. Cell. Endocrinol. 147, 85-92, 1999; Lim et al, Genes Dev. 13, 1561-74, 1999. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention. All references cited in this disclosure are expressly incorporated herein.

EXAMPLE 1

Methods used in the examples below

Tissue culture and transfections. The colorectal cancer cell line HCT116 (ATCC) and its derivatives were grown in 10% fetal bovine serum and 1% penicillin/streptomycin in McCoy's 5 A modified media and maintained at 37°C in 5% CO₂. Cells were transfected with LipofectAMTNE (Gibco/BRL) following the manufacturer's protocol and selected using hygromycin or geneticin at concentrations of 0.1 mg/ml and 0.5 mg/ml, respectively.

Generation of PPARδ null cell lines. The general strategy for creating the PPARδ null line has been described previously (17) and is outlined in the Results. In brief, the 5' and 3' homology arms used for constructing the targeting vectors were PCR amplified from HCT116 genomic DNA, using primers chosen from publicly available genomic sequence databases (GenBank accession # NT007193 ). The arms were cloned into vectors containing a hygromycin/thymidine kinase fusion gene that was flanked by loxP sequences. The primers used for deriving the targeting vectors and details of the construction are available from the authors upon request. Screening for homologous recombination events was performed by PCR as previously described (17) using the primer sequences 5'- GCTAGAGGTTTACGTGACCT-3' (SEQ ID NO:51) for the forward primer and 5'- TATGATACGGCTCAATGATG-3' (SEQ ID NO:52) for the reverse primer. After Cre mediated loxP excision, allele specific primers were used for further genetic identification. The above forward primer was used with the following reverse primers: 5'- CAGTCATAGCTCTGGCATCG-3' (SEQ ID NO:53) for the wild type allele and 5'- GTGGGGTATCGACAGAGTGC-3' (SEQ ID NO:54) for the deleted allele. Lox recombination was mediated by infection with an adenovirus expressing Cre protein (Ad- Cre) prepared as previously described (18). The PPARδ cDNA construct was created by inserting a hemaglutinin (HA) tagged

PPARδ cDNA from pCMV-HAHA-PPARδ (7) into a modified version of the pMK10₅₉ (pMK-ERES-NEO) vector (19). This modified vector was created by excising pMK10₅₉ with Xhol, blunting the ends and then inserting the resulting promo ter-IRES-NEO fragment into the EcoRN site of the loxP plasmid pBS246 (20). All targeted clones identified by PCR were verified by Southern blotting using 10 ug of genomic DΝA digested with the restriction enzyme Apa I and probed with a 400 bp genomic fragment lying just outside the 5' homology arm.

In vitro assays. In vitro assays for sulindac sulfide induced apoptosis were performed as previously described (21). All assays were performed in triplicate and the triplicates repeated at least twice. For colony formation assays, cells were plated at varying concentrations into T-25 tissue culture flasks, grown for one week and then stained with crystal violet. For in vitro growth assays, cells were seeded in triplicate in 12 well plates at a concentration of 3 x 10⁴ cells/well. Cells were harvested at days 2, 4 and 6 and viable cells as assessed by trypan blue exclusion were counted in a hemocytometer. Results were expressed as the mean of 3 wells +/- the standard deviation of the mean.

In vivo assays. Cells were harvested prior to inoculation and resuspended in serum free medium at a concentration of 5 x 10⁷ cells/ml. 5xl0⁶ cells (0.1ml) were then inoculated subcutaneously at the proximal dorsal midline into 3-4 week old female athymic nu/nu mice (Harlan) except for experiment #2 where inoculations were made in the right and left flanks. Tumor sizes in two dimensions were measured twice weekly, and volumes calculated as previously described using the formula (L x W²) x 0.5, where L= length and W=width. Mice were housed in barrier environments with food and water provided ad libitum.

Construction of a PPARδResponsive Reporter. The following oligonucleotides containing PPARδ and RXR recognition motifs that were identified from in vitro site-selection approach were synthesized: 5'-CTAGCGTG AGCGC-

TCACAGGTCAATTCGGTGAGCGCTCACAGGTCAATTCG-3' (SEQ ID ΝO:55) and 5'-CTAGCGAATTGACCTGTGAGCGCTCACCGAATTGACCTGTGAGCG-CTCACG-3 ' (SEQ ID NO:56). As a control, the following oligonucleotides containing a PPARα and PPARγ responsive element from the acyl-CoA oxidase promotor were also synthesized: 5'-CTAGCGGACCAGGACAAAGGTCACGTTCGGACC- AGGACAAAGGTCACGTTCG-3' (SEQ ID NO:57) and 5'-CTAG- CGAACGTGACCTTTGTCCTGGTCCGAACGTGACCTTTGTCCTGGTCCG-3' (SEQ ID NO:58). The oligonucleotide cassettes were dimerized and cloned into pBV-Luc, a luciferase reporter plasmid with very low basal activity. All constructs were verified by DNA sequencing.

Constructions of PPARδ Promotor Reporters. To identify the genomic sequence of the human PPARδ promotor, the following PCR primers were used to screen a BAG library (Research Genetics): 5'-CCTGTAGAGGTC-CATCTGCGTTC-3' (SEQ ID NO:59) and 5'-CATGCTGTGGTCCC-CCATTGAGC-3' (SEQ ID NO:60). Three independent BAC clones containing the PPARδ promotor sequence were obtained. Upon subcloning and sequencing, the genomic sequence of 3.1-kb immediately upstream of the first exon was determined (GenBank Accession # ). For the construction of PPARδ promotor reporters, corresponding restriction fragments

(illustrated in FIG. 2A) were subcloned into pBV-Luc. The following primer pair was used to PCR amplify the mutant NP fragment:

5'-CTAGCTAGCGAGGGTGCATCGTCAATGTTTTGTGTGGGAAG-3' (SEQ ID NO:61) and 5'-CCGGAATTCTAGGGACGATGACGATGAACAAAGCTTGA-CTC-3' (SEQ ID NO:62). The following oligonucleotide pairs were used for dimerization to construct corresponding reporters in pBV-Luc: 5'-CTAGCATGTCTTTGTAC-

TCGATGTCTTTGTACTCG-3' (SEQ ID NO:63) and 5'-CTAGCGAGTACAAAGAC- ATCGAGTACAAAGACATG-3' (SEQ ID NO:64) for p4XTREl-Luc; 5'-CTAGCATGTCTTTGGCCTCGATGTC-TTTGGCCTCG-3' (SEQ ID NO:65) and 5'-CTAGCGAGGCCAAAGACATCGAGGC-CAAAGACATG-3' (SEQ ID NO:66) for p4XmTREl-Luc; 5'-CTAGCTTGGCTTTCATCTGATTGGCTTTCATCTGAG-3' (SEQ ID NO:67) and 5'-CTAGCTCAGATGAAAGCCAATCAGATGAAAGCCAAG-3' (SEQ ID NO:68) for p4XTRE2-Luc; and 5'-CTAGCTTGGCTTTCGCCTGATTGGCTTTCG- CCTGAG-3' (SEQ LD NO:69) and 5'-CTAGCTCAGGCGAAAGCCAATCAGGC- GAAAGCCAAG-3 ' (SEQ ID NO:70) for p4XmTRE2-Luc.

Transfections and Reporter Assays. Exponentially growing cells were seeded to 12-well tissue culture plates and each assay was carried out in triplicate. Reporter plasmid, effector plasmid and β-gal control plasmid were transfected into cells using LipofectAmine (Life Technologies). Twenty-four hours after transfection, cells were lysed and collected for assays of luciferase activity using Promega's Luciferase Assay System. EXAMPLE 2

Definition of PPARδResponsive Elements (DRE)

Maximum DNA binding and activation is achieved through heterodimerization between a PPAR protein and RXR, though each protein alone can bind to its cognate recognition element, probably as a homodimer (Gearing et al., 1993; Iseemann et al., 1993). Accordingly, the prototypic PPAR response element ACO from the acyl-CoA oxidase gene promotor contains two copies of the core binding sequence AGGTCA separated by one base pair (Juge-Aubry et al, 1997; Mangelsdorf, 1995; Lemberger et al, 1996; Tugwood et al, 1992). PPARα and PPARγ bind this consensus efficiently whereas PPARδ does not (see below). To define a PPARδ responsive element, we performed in vitro binding site selection for both PPARδ and RXR. Analysis of 28 binding sites selected with a GST fusion protein containing the DNA binding domain of RXR identified (A/G)GGTCA as the core consensus for RXR (FIG. 6A). This sequence matches previously described RXR binding sites. A similar selection was performed with a GST fusion protein containing the putative DNA binding domain of PPARδ. Analysis of 20 sites identified through this selection revealed a novel binding consensus (CGCTCAC) which was distinct from the previously defined PPARα γ consensus (FIG. 6B).

We predicted that the combination of the PPARδ and RXR consensus sequences should form an efficient responsive element for PPARδ/RXR heterodimers in vivo and also create a PPARδ-binding element in vitro. To test these predictions, we first generated an oligonucleotide containing a putative PPARδ responsive element (DRE, 5'-CGCTCACAGGTCA-3') (SEQ ID NO:71) by joining the PPARδ and RXR consensus binding sites. GEMS A analysis of DRE revealed strong binding to PPARδ but not to PPARα or PPARγ (FIG. 6C). In contrast, the prototypic PPAR responsive element ACO (5'-AGGACAAAGGTCA-3') (SEQ ID NO:72) bound PPARα and PPARγ but not PPARδ (FIG. 6C). RXR demonstrated weaker binding to both responsive elements.

To test the specificity of these response elements in cells, we constructed luciferase reporters containing either the DRE or ACO elements. As expected, transfection of 293 cells with PPARδ resulted in strong activation of the DRE reporter but not the ACO reporter (FIG. 6D). In contrast, expression of PPARγ in 293 cells resulted in activation of the ACO reporter but not the DRE reporter (FIG. 6D). The above results indicate that DRE represents an effective and specific reporter of PPARδ function.

EXAMPLE 3 Generation ofPPARδnull cells

To explore the function of PPARδ in human cancer cells in a rigorous fashion, we chose to disrupt the endogenous gene by targeted homologous recombination in a human colorectal cancer cell line. The recent creation of the PPARδ null mouse had demonstrated that, similar to the other two PPAR isotypes, PPARδ is involved in lipid metabolism (22). However, PPARδ -/- mice were only generated with a Mendelian pattern of inheritance after backcrossing, suggesting that its absence may be lethal in certain mouse strains. In addition, PPARδ null mice are smaller than their control littermates and, as previously stated, PPARδ is over expressed in colorectal cancers. These results suggested that PPARδ null cancer cells may not be viable. We therefore developed a rescue strategy that enabled us to conditionally express a PPARδ cDNA prior to deletion of the second allele. This system allowed us to generate a PPARδ -/- line without concern that we had selected for a viable phenotype by clonal selection.

The HCT116 cell line chosen for these experiments contains a mutated β-catenin gene resulting in increased β-catenin/TCF-4 transcriptional activity and accordingly expresses significant amounts of PPARδ. The first allele of PPARδ was disrupted by homologous recombination using the targeting vector PKOl depicted in FIG. 1A. Recombination with PKOl was predicted to result in deletion of 11 kb of the PPARδ gene, including the entire DNA binding domain. Transfection of HCT116 with this vector and selection with hygromycin resulted in one successful recombination event among 3000 individual clones screened. This successful recombination event was confirmed by Southern blot analysis (FIG. 2). This PPARδ +/- clone was then used to generate PPARδ -/- cells using a second round of PKOl mediated recombination. In order to use the same PKOl vector in the PPARδ +/- cells, it was necessary to restore hygromycin sensitivity. This was achieved by infection with an adenovirus (Ad-Cre) expressing the Cre protein that mediated excision of the hygromycin resistance gene by virtue of the flanking loxP sequences. Multiple clones were recovered and all demonstrated loss of hygromycin resistance and excision of the hygromycin gene by Southern blot analysis. One such clone was selected for a second round of PKOl mediated recombination, as described below.

After screening approximately 5-6000 clones and failing to detect a successful recombination, we considered the possibility that complete loss of PPARδ might be lethal or severely growth inhibitory. Indeed, complete disruption of the PPARδ gene in mice can result in decreased embryo viability (22). We therefore devised a rescue strategy. In brief, the vector RV, which constitutively expressed an HA-tagged PPARδ that could be subsequently removed by Cre mediated recombination (FIG. IB), was transfected into the HCT116 PPARδ +/- cells. PPARδ +/- clones with the RV (PPARδ +/-/RV) were then selected for their resistance to geneticin, which ensured expression of the PPARδ cDNA due to the modified IRES sequence (19) separating the PPARδ cDNA and the geneticin resistance gene (FIG. IB). Expression of the HA- PPARδ protein in PPARδ +/-/RV clones was demonstrated by Western blotting using an anti-HA antibody (data not shown). Transfection of a representative PPARδ +/-/RV clone with the PKOl vector resulted in one successful recombination event after screening approximately 1000 clones. Southern blotting confirmed that homologous recombination had deleted the remaining endogenous PPARδ allele resulting in the line PPARδ -/-/RV, as shown in FIG. 2.

EXAMPLE 4 PPARδ null cell lines are viable in vitro

The PPARδ -/-/RV cell line was then used to test whether deletion of PPARδ was lethal or severely growth inhibitory. The PPARδ -/-/RV cells could be easily rendered PPAR null by Cre mediated excision after infection with Ad-Cre. As a control for nonspecific effects, a sister PPARδ +/-/RV clone was similarly infected with Ad-Cre. Surprisingly, clones that had excised the PPARδ RV were recovered in equal numbers from both the PPARδ -/-/RV and PPARδ +/-/RV clones, indicating that PPARδ is not required for in vitro growth. Excision of the PPARδ cDNA was confirmed by both PCR and western blot analysis (data not shown). There were no obvious differences between the growth properties between the two groups of recovered clones in vitro, though both PPARδ +/- and PPARδ -/- clones grew slightly slower than wild type HCT116 cells (see below). To rule out the possibility of clonal variability, additional PPARδ null cell lines were sought. Unfortunately, the low recombination frequency of the targeting vector significantly hampered our ability to generate more clones. To circumvent this problem, a second generation targeting vector (PKO2) was constructed that had a longer 5' homology arm (1.8 kb vs 1.0 kb) and deleted only the first coding exon of the PPARδ gene encompassing 200 bp as shown in FIG. 1A. Preliminary experiments in our laboratory suggested that decreasing the size of the deleted region might increase the recombination frequency of targeted homologous recombinations. We therefore hoped that recombination rates with PKO2 might be superior to PKOl. Indeed, transfection of wild type HCT116 resulted in 11 successful recombinations among 200 clones tested, representing an increase in targeting efficiency of ~150-fold. Likewise, transfection of PPARδ +/-/RN resulted in two successful recombinations among 200 clones tested. The two PPARδ -/-/RN clones were confirmed by Southern blotting and subjected to infection with Ad-Cre to generate PPARδ null cells. As with the original null clone, viable clones could be recovered at similar frequencies, demonstrating that deletion of the PPARδ gene was not lethal.

EXAMPLE 5

PPARδ is not required for sulindac mediated apoptosis

We had previously suggested that inhibition of PPARδ by NSAIDs might contribute to their chemopreventive effects. One potential in vitro surrogate of NSAID chemoprevention is the ability to induce apoptosis. Consistent with a possible role of PPARδ, we have previously demonstrated that over expression of PPARδ could partially rescue cells from sulindac induced apoptosis (7). To determine whether PPARδ is required for sulindac mediated apoptosis, wild type HCT116, two independently derived PPARδ -/- clones and a sister PPARδ +/- heterozygote clone were analyzed for their response to sulindac. Cells were treated with sulindac at various concentrations, and then stained with crystal violet to assess the number of surviving cells after 72 hours. There was no appreciable difference in cell viability between these four cell lines (FIG. 3) suggesting that PPARδ is not required for NSAID mediated apoptosis. EXAMPLE 6

PPARδ is required for efficient tumorigenicity

Although the above data conclusively demonstrated that PPARδ is not required for in vitro cell growth, we questioned whether the absence of this receptor had any effect on tumorgenicity. To address this hypothesis, wild type HCT116, PPARδ +/-, and PPARδ -/- cells were injected subcutaneously and grown as xenografts in nude mice. There was a dramatic difference in tumor establishment and growth of the PPARδ -/- cells compared to wild type HCT116 and the PPARδ +/- cells. This in vivo growth deficiency was observed in five independent experiments (Table 1). The "Frequency" column indicates the fraction of inoculation sites in which a tumor grew. The "All Tumors" column indicates the average volume (mm³) +/- standard error of tumors for all inoculation sites, whereas the "Established Tumors" column indicates the average volume (mm³) +/- standard error for only those tumors that were clearly detectable on the date of the final measurement, as indicated in parentheses next to the experiment number. NA indicates not applicable. While there was no appreciable difference in tumor establishment or growth between wild type HCT116 and the PPARδ +/- cells, both of the PPARδ -/- cell lines yielded fewer tumors per mouse compared with wild type and PPARδ +/- cells (Table 1 and FIG. 4). In addition, the few tumors that did arise in the mice were smaller than their wild type or PPARδ +/- control counterparts (Table 1 and examples in FIG. 4A). It is important to note that the two PPARδ null cell lines used in these experiments were independently derived. The fact that both lines were defective in growth in nude mice indicated that this in vivo phenotype was dependent on disruption of the PPARδ gene and not the result of clonal variability. To further assess this issue, a total of four additional independently derived PPARδ null clones were tested for their in vivo tumorgenicity, and all displayed similar defects in tumor growth relative to the wild type and PPAR (+/-) cells (data not shown). The in vivo growth defects of the PPARδ null cells did not simply reflect general alterations in growth, as all lines grew well in culture. Quantitative analysis demonstrated that the PPARδ +/- and PPARδ -/- cells were indistinguishable in their in vitro growth properties, though parental HCT116 cells with completely intact PPARδ genes generally grew somewhat better than the modified cells (FIGS. 5A and 5B). The absence of PPARδ clearly affects tumorgenicity in the HCT116 cell line. Because the APC pathway is mutated with high frequency in colon cancer and over expression of PPARδ has been observed in many colorectal cancers, it seems likely that increased expression of PPARδ generally contributes to the neoplastic process. Furthermore, the fact that PPARδ is a transcription factor suggests that such growth promoting effects are directly related to expression and/or repression of target genes. As such, a comprehensive analysis of gene expression using the PPARδ -/- cells and appropriate controls may enable identification of important growth regulatory genes.

To our knowledge, there is only one gene whose disruption in a colorectal cancer cell line has been shown to limit tumorigenicity. Sasazuki and colleagues showed that disruption of the mutant K-RAS gene in HCT116 cells resulted in a phenotype very similar to that observed in the PPARδ null cells: continued growth in vitro but poor growth in vivo (27). Such experiments provide critical information for defining appropriate targets for therapy.

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Table 1 - In vivo growth of PPARδ null cells

Frequencv All Tumors Establisl hied Tumors

Exp. #1 (28 days)

PPARδ +/+ 2/2 1218 +/- 1095 1218 +/- 1095

PPARδ +/- 2/2 987 +/- 49 987 +/- 49

PPARδ -/- Clone 1 1/2 2 +1- 2 4 +/- NA

Exp. #2 (30 days)

PPARδ +/+ 4/4 1306 +/- 680 1306 +/- 680

PPARδ +/- 4/4 2333 +/- 803 2333 +/- 803

PPARδ -/- Clone 1 4/4 374 +/- 46 374 +/- 46

Exp. #3 (33 days)

PPARδ +/+ 10/10 1617 +/- 248 1617 +/- 248

PPARδ -/- Clone 1 4/10 271 +/- 107 609 +/- 90

Exp. #4 (34 days)

PPARδ +/+ 5/5 1922 +/- 823 1922 +/- 823

PPARδ -/- Clone 2 2/5 86 +/- 64 214 +/- 1 15

Exp. #5 (34 days)

PPARδ +/+ 6/6 673 +/- 80 673 +/- 80

PPARδ +/- 5/5 732 +/- 164 732 +/- 164

PPARδ -/- Clone 1 2/5 56 +/- 48 141 +/- 66

PPARδ -/- Clone 2 1/5 49 +/- 49 243 +/- NA

Claims

1. A homozygous PPARδ gene-defective cell line.

2. The cell line of claim 1 which comprises human cells.

3. The cell line of claim 1 which comprises colon cells.

4. The cell line of claim 1 which comprises tumor cells.

5. The cell line of claim 1 which comprises human colon tumor cells.

6. The cell line of claim 1 which is a PPARδ mutant of HCT116 cells.

7. The cell line of claim 1 wherein cells of the cell line comprise a reporter construct comprising a reporter gene which encodes an assayable product, a minimal promoter upstream from and regulating transcription of the reporter gene, and at least one copy of a PPARδ binding element upstream of the minimal promoter.

8. The cell line of claim 7 wherein the reporter construct further comprises an RXR binding element upstream of the minimal promoter.

9. A pair of isogenic cell lines wherein cells of a first cell line are homozygous PPARδ gene-defective and cells of a second cell line are homozygous PPARδ gene wild-type.

10. The pair of claim 9 wherein the cell lines comprise human cells.

11. The pair of claim 9 wherein the cell lines comprise colon cells.

12. The pair of claim 9 wherein the cell lines comprise tumor cells.

13. The pair of claim 9 wherein the cell lines comprise human colon tumor cells.

14. The pair of claim 9 wherein the second cell line is HCT116.

15. The pair of claim 9 wherein cells of the first and second cell lines comprise a reporter construct comprising a reporter gene which encodes an assayable product, a minimal promoter upstream from and regulating transcription of the reporter gene, and at least one copy of a PPARδ binding element upstream of the minimal promoter.

16. The pair of claim 9 wherein the reporter construct further comprises an RXR binding element upstream of the minimal promoter.

17. The pair of claim 9 wherein the cell lines are in a single, divided container.

18. A method of identifying a test compound as a PPARδ-specific antagonist or agonist, comprising the steps of: contacting a first and a second isogenic cell with a test compound, wherein the first cell is a homozygous PPARδ gene-defective cell and wherein the second cell is a homozygous PPARδ gene wild-type cell; assessing rate of division of the first and second cells; and identifying a test compound that reduces the rate of division of the second cell relative to the first cell as a PPARδ-specific antagonist or identifying a test compound that enhances the rate of division of the second cell relative to the first cell as a PPARδ-specific agonist.

19. The method of claim 18 wherein the test compound is a non-steroidal anti- inflammatory drug.

20. The method of claim 18 wherein the first and second cells are human cells.

21. The method of claim 18 wherein the first and second cells are colon cells.

22. The method of claim 18 wherein the first and second cells are tumor cells.

23. The method of claim 18 wherein the first and second cells are human colon tumor cells.

24. The method of claim 18 wherein the second cell is an HCT116 cell.

25. A method of identifying a test compound as a PPARδ-specific agonist or antagonist, comprising the steps of: contacting a first and a second isogenic cell with a test compound, wherein each of the cells contains a reporter construct comprising (a) a reporter gene which encodes an assayable product, (b) a minimal promoter upstream from and regulating transcription of the reporter gene, and (c) at least one copy of a PPARδ binding element upstream of the minimal promoter, wherein the first cell is homozygous PPARδ gene-defective and wherein the second cell is homozygous PPARδ gene wild-type; determining expression of the reporter gene; and identifying a test compound that decreases the amount of expression of the reporter gene in the second cell relative to the first cell as a PPARδ-specific antagonist or identifying a test compound which increases the amount of expression of the reporter gene in the second cell relative to the first cell as a PPARδ-specific agonist.

26. The method of claim 25 wherein the reporter gene constuct further comprises an RXR binding element upstream of the minimal promoter.

27. The method of claim 25 wherein the first and second isogenic cells are contacted with the test compound in vitro.

28. The method of claim 25 wherein the first and second isogenic cells are contacted with the test compound in a host animal.

29. The method of claim 25 wherein the test compound is a non-steroidal anti- inflammatory drug.

30. The method of claim 25 wherein the pair of isogenic cells are human cells.

31. The method of claim 25 wherein the pair of isogenic cells are colon cells.

32. The method of claim 25 wherein the pair of isogenic cells are tumor cells.

33. The method of claim 25 wherein the pair of isogenic cells are human colon tumor cells.

34. The method of claim 25 wherein the second cell is an HCT116 cell.

35. A method of identifying a test compound as a PPARδ-specific antagonist or agonist, comprising the steps of: administering a test compound to a first and second experimental animal; providing the first and second animals with xenografts, wherein the first animal is provided with a xenograft comprising homozygous PPARδ gene-defective tumor cells and the second animal is provided with a xenograft comprising PPARδ gene wild-type tumor cells, wherein the PPARδ gene-defective and the PPARδ gene wild-type cells are isogenic; and assessing tumorigenicity of the PPARδ gene-defective and the PPARδ gene wild-type cells, wherein a test compound that reduces the tumorigenicity of the PPARδ gene wild-type cells relative to the PPARδ gene-defective cells is identified as a PPARδ-specific antagonist and wherein a test compound that enhances the tumorigenicity of the PPARδ gene wild-type cells relative to the PPARδ gene-defective cells is identified as a PPARδ-specific agonist.

36. The method of claim 35 wherein the PPARδ gene wild-type and the PPARδ gene- defective tumor cells are human cells.

37. The method of claim 35 wherein the PPARδ gene wild-type and the PPARδ gene- defective tumor cells are colon cells.

38. The method of claim 35 wherein the PPARδ gene wild-type and the PPARδ gene- defective tumor cells are human colon tumor cells.

39. The method of claim 35 wherein the PPARδ gene wild-type tumor cells are HCT116 cells.

40. The method of claim 35 wherein the test compound is a non-steroidal anti- inflammatory drug.

41. The method of claim 35 wherein tumorigenicity is assessed by counting the number of tumors formed by the xenografts.

42. The method of claim 35 wherein tumorigenicity is assessed by measuring the rate of tumor formation by the xenografts.

43. The method of claim 35 wherein tumorigenicity is assessed by measuring the size of tumors formed by the xenografts.

44. The method of claim 35 wherein the xenografts are provided before administration of the test compound.