JP2005514906A - LCEs as p53 pathway modifiers and methods of use - Google Patents

Abstract

  Human LCE genes have been identified as modulators of the p53 pathway and are therefore therapeutic targets for diseases associated with defective p53 function. Methods are provided for identifying modulators of p53 comprising screening to look for agents that modulate the activity of LCE.

Description

(Reference to related applications)
This application is based on US Provisional Application No. 60 / 296,076, filed June 5, 2001, US Provisional Application No. 60 / 328,605, filed October 10, 2001, February 15, 2002. US Patent Provisional Application No. 60 / 357,253, filed, and US Patent Provisional Application No. 60 / 361,196, filed March 1, 2002, are claimed. The content of the prior application is incorporated herein in its entirety.

(Background of the Invention)
The p53 gene has been mutated across over 50 different human cancers, including familial and spontaneous cancers, and is considered the most widely mutated gene among human cancers (Zambetti and Levine FASEB, 1993, 7: 855-865; Hollstein et al., Nucleic Acids Res., 1994, 22: 3551-3555). More than 90% of mutations in the p53 gene are missense mutations that alter single amino acids that inactivate p53 function. Abnormal forms of human p53 are associated with poor prognosis, more active tumors, metastasis, and short-term survival (Mitsudomi et al., Clin Cancer Res, October 2000, 6 (10): 4055-63; Koshland, Science, 1993, 262: 1953).

  Human p53 protein normally functions as a central integrator of signals including DNA damage, hypoxia, nucleotide deficiency, and oncogene activation (Prives, Cell, 1998, 95: 5-8). In response to these signals, p53 protein levels are greatly elevated, so that accumulated p53 activates cell cycle arrest or cell death depending on the nature and intensity of these signals. Indeed, multiple lines of experimental evidence pointed to the major role of p53 as a tumor suppressor (Levine, Cell, 1997, 88: 323-331). For example, homozygous p53 “knockout” mice, although normal in development, showed a near 100% incidence of new tissue formation during the first year of life (Donehower et al., Nature, 1992, 356: 215). ~ 221).

  Although the biochemical mechanism and pathway by which p53 functions in normal and cancer cells is not fully understood, one clearly important aspect of p53 function is its activity as a gene-specific transcriptional activator. Some genes with known p53 response elements have a role in the regulation of either cell cycle or cell death, including GADD45, p21 / Waf1 / Cip1, cyclin G, Bax, IGF-BP3, and MDM2. There are several that have been characterized (Levine, Cell, 1997, 88: 323-331).

  Some important organs (eg lung, kidney, lymphatic system and vasculature) consist of a complex network of tubular structures that contribute to the transport and exchange of fluids, gases, nutrients and waste products. The formation of a branched complex network occurs by an evolutionarily conserved branch morphogenesis process, and continuous branching occurs by budding, pruning and remodeling of the network. During human embryogenesis, blood vessels develop by two processes: angiogenesis in which endothelial cells are born from the progenitor cell type and angiogenesis in which new capillaries sprouting from existing vessels.

  Branching morphogenesis involves many cellular processes including proliferation, survival / apoptosis, migration, invasion, adhesion, assembly and substrate remodeling. Many types of cells contribute to branching morphogenesis, including endothelial cells, epithelial and smooth muscle cells, and mononuclear cells. Genetic pathways that regulate the branching process function both in branching tissues and in other cells, for example, some mononuclear cells may not participate directly in the formation of branching structures, but angiogenesis Response can be facilitated.

  Increasing levels of angiogenesis is central to several human disease pathologies, including rheumatoid arthritis and diabetic retinopathy, and is particularly important for solid cancer growth, maintenance and metastasis (more details) Liotta LA et al., 1991, Cell 64: 327-336; Folkman J., 1995, Nature Medicine 1: 27-31; Hanahan D and Folkman J, 1996, Cell 86: 353-364). Angiogenesis disorders are found in other human diseases including heart disease, stroke, infertility, ulcers and scleroderma.

  The transition from dormancy to active angiogenesis involves regulating the balance of stimulators and inhibitors of angiogenesis. Depending on the pathological environment, there may be an imbalance between local inhibitory control and angiogenesis inducers, resulting in excessive angiogenesis, or under other pathological conditions, an imbalance may result in angiogenesis deficiency . The delicate balance between pro and anti-angiogenic factors is controlled by a complex interaction between extracellular matrix, endothelial cells, smooth muscle cells and various other cell types, and environmental factors such as oxygen demand in tissues. The

  Most of the well-known angiogenic genes, their biochemical activity, and organization into signaling pathways are similar during angiogenesis in humans, mice and zebrafish and during branching morphogenesis of Drosophila trachea in use. Thus, Drosophila tracheal development and zebrafish vascular development provide a useful model for the study of mammalian angiogenesis (Metzger RJ, Krasnow MA. Science. 1999. 284: 1635-9; Roman BL And Weinstein BM. Bioessays 2000, 22: 882-93).

  In mammals, many of the newly synthesized fatty acids have a chain length of 16-18 carbons. These long chain fatty acids are over 90% of the total fatty acids present in the cell. They are an important component of the membrane and the animal's maximum energy storage reservoir. The highest rate of new fatty acid synthesis occurs in the liver, which converts excess glucose to fatty acids for storage and transport. Glycolysis converts glucose to pyruvate, which is converted to citrate by mitochondria and transported to the cytosol. Cytosolic ATP citrate lyase produces acetyl CoA, a precursor of fatty acids and cholesterol. Acetyl CoA is carboxylated by acetyl CoA carboxylase (ACC) to form malonyl CoA. The multifunctional enzyme fatty acid synthase (FAS) uses malonyl CoA, acetyl CoA, and NADPH to extend fatty acids by two carbons. The major end product of FAS in rodents is palmitic acid, which has 16 carbons and is shown at 16: 0. This high proportion of palmitic acid is then converted to stearic acid.

  At the molecular level, fatty acid elongases are the most extensively studied in yeast genetic research. Yeast ELO1 extends fatty acids from C14 to C16 and is labeled as a long chain fatty acid elongase (Toke DA et al., 1996, J Biol Chem 271: 18413-18422). The ELO2 and ELO3 genes encode very long chain elongases that produce 24-26 carbon fatty acids (Oh CS et al., 1997, J Biol Chem 272: 17376-17384). The mouse gene Cig30 encodes a mouse version of ELO2, and Ssc1 encodes an ortholog of ELO3. They are both very long chain elongases (Tvrdik P et al., 2000, J Cell Biol 149: 707-718). CIG30 and other fatty acid elongases have been used for sphingolipid biosynthesis. In certain cells, sphingosine kinase is exported to the extracellular space where it is phosphorylated to produce phospho-sphingosine (S1P) (Hla et al., Science, 2001, 294: 1875-8). S1P is a powerful second messenger that binds to EDG1 GPCR and mediates migration, survival, phenotyping and proliferation. S1P and EDG1 have been shown to be involved in endothelial cell migration and vascular maturation in vivo (Ancellin et al., J Biol Chem 2002, 277: 6667-75).

  A mouse long-chain fatty acyl elongase (LCE) has been identified that shares sequence identity with the previously identified very long-chain fatty acid elongase (elongase). LCE mRNA is highly expressed in liver and adipose tissue and is thought to catalyze the rate-limiting condensation step in the addition of 2-carboxylic units to C12-C16 fatty acids (Moon YA et al., 2001, J Biol Chem 276: 45358-66). ). One of the human LCE genes, ELOVL4, has been identified as a disease gene-dominant Stargardt-like macular dystrophy (STGD3)-related to inheritance in the form of macular formation characterized by visual acuity, macular atrophy and extensive fundus mottling. Zhang et al., Nat Genet 2001, 27: 89-93). All affected members of the five related families have a 5 bp deletion of the gene (so-called 797-801delAACTT).

Manipulating the genome of a model organism, such as Drosophila, provides a powerful means of analyzing biochemical processes that have a direct relevance to complex vertebrates with a number of significant evolutionary conservation. The high level of conservation of genes and pathways, the high similarity of cellular processes, and the conservation of gene functions between these model organisms and mammals have led to the involvement of new genes in specific pathways and Identification of its function in such model organisms can directly contribute to understanding correlation pathways in mammals and how to regulate them (eg, Mechler BM et al., 1985, EMBO J, 4 Gateff E., 1982, Adv. Cancer Res., 37: 33-74; Watson KL. Et al., 1994, J Cell Sci., 18: 19-33; Miklos GL and Rubin GM., 1996, Cell, 86: 521-529; Wassarman DA et al., 1995, Curr Opin Gen Dev, 5: 44-50; Booth DR., 1999, Cancer Metastasis Rev., 18: 261-284). For example, genetic screening can be performed in a spinal model organism that has underexpression (eg, knockout) or overexpression (called “genetic entry point”) of a gene that results in a visible phenotype . Additional genes are mutated randomly or in a targeted manner. If a mutation in a gene changes the initial phenotype caused by the genetic entry point, the gene is identified as a “modifier” that is involved in the same or overlapping pathway as the genetic entry point. If the genetic entry point is an ortholog of a human gene that is associated with a disease pathway such as p53, a modifier gene can be identified that can be an attractive candidate target for a new therapeutic agent.

  All references cited herein, including referenced Genbank identification numbers and website references, are incorporated herein in their entirety.

(Summary of Invention)
The inventors have discovered a gene that alters the p53 pathway in Drosophila and identified its ortholog in humans, hereinafter referred to as LCE. The present invention provides methods for identifying candidate therapeutic agents that can be used to treat diseases associated with defective p53 function utilizing these p53 modifier genes and polypeptides. Preferred LCE modulating agents specifically bind to LCE polypeptides and restore p53 function. Other preferred LCE modulators are nucleic acid modulators such as antisense oligomers, or RNAi that suppress LCE gene expression or product activity by binding to and inhibiting the corresponding nucleic acid (ie, DNA or mRNA), for example. .

  Modulators specific for LCE can be assessed by any convenient in vitro or in vivo assay for molecular interaction with an LCE polypeptide or nucleic acid. In one embodiment, candidate p53 modulating agents are tested using an assay system comprising an LCE polypeptide or nucleic acid. Candidate agents that cause a change in the activity of the assay system relative to the control are identified as candidate p53 modulating agents. The assay system may be cell based or cell free. LCE modulators include LCE-related proteins (eg, dominant negative mutants and biotherapeutics); antibodies specific for LCE; antisense oligomers and other nucleic acid modulators specific for LCE; Chemical agents that compete with the LCE binding target are included. In one particular embodiment, binding assays are used to identify small molecule modulators. In certain embodiments, the screening assay is selected from an apoptosis assay, a cell proliferation assay, an angiogenesis assay, and a hypoxia induction assay.

  In another embodiment, a second assay system that detects changes in the p53 pathway, such as changes in angiogenesis, cell death, or cell proliferation caused by initially identified candidate agents or agents derived from the first agent Are used to further test candidate p53 pathway modulators. Cultured cells or non-human animals can be used for the second assay system. In certain embodiments, the secondary assay system is an animal previously determined to have a disease or disorder associated with the p53 pathway, such as an angiogenesis, cell death, or cell proliferation disease (eg, cancer). Use non-human animals, including

  The invention further provides a method of modulating the p53 pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds to an LCE polypeptide or nucleic acid. The agent can be a small molecule modulator, a nucleic acid modulator, or an antibody and can be administered to a mammal that has been previously determined to have a pathology associated with the p53 pathway.

  The present invention further provides methods for identifying candidate branched morphogenesis regulators and for modulating branched morphogenesis in mammalian cells using LCE.

(Detailed description of the invention)
A genetic screen was designed to identify modifiers of the p53 pathway in Drosophila that had overexpressed p53 in the cage (Ollmann M et al., Cell 2000, 101: 91-101). The boldspot gene has been identified as a modifier of the p53 pathway. Thus, LCE genes (ie, nucleic acids and polypeptides) that are vertebrate orthologs of these modifiers, preferably human orthologs, (ie, nucleic acids and polypeptides) are attractive drug targets in the treatment of pathologies associated with defective p53 signaling pathways such as cancer. .

  Furthermore, LCE (ELOVL4) involved in branching morphogenesis, particularly angiogenesis and angiogenesis, was identified. This will be further described in the examples. Thus, ELOVL4 is an interesting target reagent for the treatment of pathologies related to branching morphogenesis, including the treatment of tumors that grow in association with increased angiogenesis.

  The present invention provides in vitro and in vivo methods for evaluating LCE function. Modulation of LCE or its corresponding binding partner is useful for understanding the relationship between the p53 pathway and its members in normal conditions and pathologies, and developing diagnostic methods and treatment modalities for p53-related pathologies. Modulation of ELOVL4 or its binding partner can further elucidate the process of branching morphogenesis and the relationship between branching morphogenesis and the p53 pathway and develop diagnostic methods and treatment modalities for pathological conditions associated with branching morphogenesis Useful. As used herein, the term branched morphogenesis encompasses the myriad cellular processes associated with branched networks, including proliferation, survival / apoptosis, migration, invasion, adhesion, aggregation and matrix remodeling. As used herein, pathological conditions associated with branching morphogenesis include pathologies where branching morphogenesis contributes to the maintenance of a healthy state, as well as those that can change the course through modulation of branching morphogenesis. To do.

  LCEs that act by directly or indirectly affecting LCE function, such as enzymatic (eg, catalytic) activity or binding activity, to inhibit or enhance LCE expression using the methods provided herein. Modulators can be identified. LCE modulators are useful in diagnosis, therapy, and pharmaceutical development.

Nucleic acids and polypeptides of the present invention The sequences related to LCE nucleic acids and polypeptides that can be used in the present invention are GI # 104444344 (SEQ ID NO: 1), GI # 13129087 (SEQ ID NO: 3), GI # 10440044 SEQ ID NO: 4), GI # 120404402 (SEQ ID NO: 5), GI # 122232378 (SEQ ID NO: 6), GI # 18576451 (SEQ ID NO: 8), polypeptide is GI # 104444345 (SEQ ID NO: 9), GI # 13129088 (SEQ ID NO: 9) 11), GI # 10440045 (SEQ ID NO: 12), GI # 12040443 (SEQ ID NO: 13), GI # 122232379 (SEQ ID NO: 14), and GI # 17445417 (SEQ ID NO: 16) are disclosed in Genbank (Genbank identification ( GI) See by number See). In addition, the nucleic acid sequences of SEQ ID NOs: 2 and 7 and the polypeptide sequences of SEQ ID NOs: 10 and 15 can be used in the present invention.

  LCE is a fatty acid elongase protein having a GNS1 / SUR4 domain. The term “LCE polypeptide” refers to a full length LCE protein or a functionally active fragment or derivative thereof. A “functionally active” LCE fragment or derivative has one or more functional activities associated with the full-length wild-type LCE protein, such as antigenic or immunogenic activity, enzymatic activity, ability to bind to natural cellular substrates, or Show multiple. The functional activity of LCE proteins, derivatives and fragments can be determined by various methods well known to those skilled in the art (Current Protocols in Protein Science, 1998, Coligan et al., John Wiley & Sons, Inc., Somerset, NJ) and below It can be assayed as further described. For the purposes of the present invention, functionally active fragments also include fragments comprising one or more LCE structural domains such as reductase domains and binding domains. Protein domains can be identified using the PFAM program (Bateman A. et al., Nucleic Acids Res, 1999, 27: 260-2; http://pfam.wustl.edu). For example, the GNS1 / SUR4 domain (PFAM01151) of LCE of GI # 104444345, GI # 13129088, GI # 122232379 and GI # 17445417 (SEQ ID NOs: 9, 11, 14 and 16 respectively) is generally amino acid residues 1-235, 10-265, 9-289, and 55-270. Methods for obtaining LCE polypeptides are further described below. In some embodiments, preferred fragments are at least 25 contiguous sequences of any one of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, or 16 (LCE) that are functionally active. Domain-containing fragment comprising at least 50, more preferably 75, most preferably 100 consecutive amino acids. In a further preferred embodiment, this fragment comprises the entire GNS1 / SUR4 (functionally active) domain.

  The term “LCE nucleic acid” refers to a DNA or RNA molecule that encodes an LCE polypeptide. Preferably, the LCE polypeptide or nucleic acid or fragment thereof is of human origin, but at least 70% sequence identity with LCE, preferably at least 80%, more preferably 85%, even more preferably 90%, most preferably It may be an ortholog or derivative thereof having at least 95% sequence identity. Usually, different species of orthologs retain the same function due to the presence and / or three-dimensional structure of one or more protein motifs. In general, orthologs are typically identified by sequence homology analysis, such as BLAST analysis, using protein bait sequencing. If the best-matched sequence of forward BLAST results retrieves the original query sequence of reverse BLAST, designate the sequence as a potential ortholog (Huynen MA and Bork P, Proc Natl Acad Sci, 1998, 95: 5849 -5856; Huynen MA et al., Genome Research, 2000, 10: 1204-1210). Using a program for multiple sequence alignment, such as CLUSTAL (Thompson JD et al., 1994, Nucleic Acids Res 22: 4673-4680), highlights conserved regions and / or residues of orthologous proteins, and a phylogenetic tree May be created. In a phylogenetic tree representing multiple types of multiple homologous sequences (eg, those extracted by BLAST analysis), the two orthologous sequences appear closest in the phylogenetic tree with respect to all the other two sequences. Potential orthologs may be identified by structural threading or other methods of protein folding (eg, using ProCeryon (Bioscience, Salzburg, Austria)). In evolution, when gene duplication occurs following speciation, a single species of a single species, such as Drosophila, may correspond to multiple genes of another species, such as humans (paralog). In this specification, the expression “ortholog” includes paralogs. “Percent (%) sequence identity” as used herein with respect to a subject sequence or a specific portion of a subject sequence is all that is necessary to align sequences and obtain maximum percent sequence identity. WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol., 1997, 215: 403-410; http://blast.wustl.edu/blast/README) defined as the percentage of nucleotides or amino acids in the sequence of candidate derivatives that are identical to the nucleotides or amino acids in the subject sequence (or a specific portion thereof) after introducing the gap created by .html). The HSP S and HSP S2 parameters are dynamic values and are determined by the program itself depending on the composition of the specific sequence and the composition of the individual databases to be searched against the target sequence. The percent identity value is determined by dividing the number of matching identical nucleotides or amino acids by the length of the sequence for which percent identity is reported. “Percent (%) amino acid sequence similarity” is determined by performing the same calculation as determining% amino acid sequence identity but including conservative amino acid substitutions in addition to the same amino acid.

  A conservative amino acid substitution is a substitution in which one amino acid is replaced with another amino acid having similar properties so that protein folding and activity are not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; compatible hydrophobic amino acids are leucine, isoleucine, methionine, and valine; compatible polar amino acids are glutamine and asparagine; interchangeable The basic amino acids are arginine, lysine and histidine, the compatible acidic amino acids are aspartic acid and glutamic acid, and the compatible small amino acids are alanine, serine, threonine, cysteine and glycine.

  Alternatively, nucleic acid sequence alignments are provided by Smith and Waterman's local homology algorithm (Smith and Waterman, 1981, Advances in Applied Mathematics, 2: 482-489; database: European Bioinformatics Institute http: // www. ebi.ac.uk/MPsrch/; Smith and Waterman, 1981, J. of Molec. Biol., 147: 195-197; Nicholas et al., 1998, `` A Tutorial on Searching Sequence Databases and Sequence Scoring Methods '' (www. .psc.edu) and references cited therein, WRPearson, 1991, Genomics, 11: 635-650). This algorithm was developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, edited by MODayhoff, 5th appendix, 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA) and normalized by Gribskov (Gribskov 1986, Nucl. Acids Res. 14 (6): 6745-6673) can be applied to amino acid sequences by using a scoring matrix. A Smith-Waterman algorithm with initial parameters can be used to score (e.g. gap open penalty 12, gap extension penalty 2). In the generated data, the “match” value reflects “sequence identity”.

  Nucleic acid molecules derived from the subject nucleic acid molecules include sequences that hybridize to any nucleic acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. Hybridization stringency can be adjusted by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Routinely used conditions are described in readily available procedures (eg, Current Protocol in Molecular Biology, Volume 1, Chapter 2.10, John Wiley & Sons, Publishers, 1994; Sambrook et al., Molecular Cloning, Cold Spring Harbor, 1989). In some embodiments, a nucleic acid molecule of the invention comprises 6 × unit strength citrate (SSC) (1 × SSC is 0.15 M NaCl, 0.015 M Na citrate, pH 7.0). Pre-hybridize the filter containing the nucleic acid at 65 ° C. for 8 hours to overnight in a solution containing 5 × Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg / ml herring sperm DNA. Performing hybridization at 65 ° C. for 18-20 hours in a solution containing 6 × SSC, 1 × Denhardt's solution, 100 μg / ml yeast tRNA and 0.05% sodium pyrophosphate; and 0.2 X A solution containing SSC and 0.1% SDS (sodium dodecyl sulfate), filled at 65 ° C for 1 hour Hybridizing to a nucleic acid molecule comprising a nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8 under stringent hybridization conditions including washing Can do.

  In other embodiments, 35% formamide, 5 × SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and Pretreat the filter containing nucleic acid for 6 hours at 40 ° C. in a solution containing 500 μg / ml denatured salmon sperm DNA; 35% formamide, 5 × SSC, 50 mM Tris-HCl (pH 7.5), In a solution containing 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, and 10% (weight / volume) dextran sulfate, Perform hybridization at 40 ° C. for 18-20 hours; then wash twice with a solution containing 2 × SSC and 0.1% SDS for 1 hour at 55 ° C. Use moderate stringency hybridization conditions, including

  Alternatively, in a solution containing 20% formamide, 5 × SSC, 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, Incubate at 37 ° C. for hours to overnight; perform hybridization for 18-20 hours in the same buffer; and wash for 1 hour at 37 ° C. with 1 × SSC for low stringency Conditions can be used.

Isolation, generation, expression, and misexpression of LCE nucleic acids and polypeptides LCE nucleic acids and polypeptides are useful for the identification and testing of agents that modulate LCE function and other uses related to LCE involvement in the p53 pathway. is there. LCE nucleic acids and their derivatives and orthologs can be obtained using any available method. Techniques for isolating a cDNA or genomic DNA sequence of interest, for example, by screening a DNA library or by using polymerase chain reaction (PCR) are well known in the art. In general, the specific use of a protein defines the details of expression, production, and purification methods. For example, generating proteins for use in screening to find modulators may require methods to preserve the specific biological activity of these proteins, but generate proteins to produce antibodies May require the structural integrity of a particular epitope. Expression of proteins to be purified for screening or antibody production may require the addition of specific tags (eg, production of fusion proteins). Overexpression of LCE proteins for assays used to assess LCE function, such as cell cycle control and involvement of hypoxic responses, requires expression in eukaryotic cell systems capable of these cellular activities May be. Methods for expressing, producing, and purifying proteins are well known in the art, and thus any suitable means can be used (eg, Higgins SJ and Hames BD, Protein Expression: A Practical Approach, Oxford University Press Inc., New York, 1999; Stanbury PF et al., Principles of Fermentation Technology, 2nd edition, Elsevier Science, New York, 1995; Doonan S, Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, New York). In a specific embodiment, the recombinant LCE is a cell line known to have defective p53 function (eg, SAOS-2 osteoblasts, particularly available from American Type Culture Collection (ATCC), Manassas, Va.). , H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT-29 and DLD-1 colon cancer cells,). This recombinant cell is used in the cell-based screening assay system of the present invention described further below.

  The nucleotide sequence encoding the LCE polypeptide can be inserted into any suitable expression vector. The necessary transcriptional and translational signals, including promoter / enhancer elements, can be derived from the native LCE gene and / or its flanking regions or can be heterologous. Mammalian cell lines infected with viruses (eg, vaccinia virus, adenovirus, etc.); insect cell lines infected with viruses (eg, baculovirus); yeast containing yeast vectors, or traited with bacteriophage, plasmid, or cosmid DNA Various host-vector expression systems such as transformed microorganisms such as bacteria can be used. Host cell systems that modulate, modify, and / or specifically process the expression of the gene product can be used.

  To detect the LCE gene product, the expression vector comprises a promoter operably linked to the nucleic acid of the LCE gene, one or more origins of replication, and one or more selectable markers (eg, thymidine kinase activity, Antibiotic resistance, etc.). Alternatively, recombinant expression vectors can be identified by assaying the expression of the LCE gene product based on the physical or functional properties of the LCE protein in an in vitro assay system (eg, an immunoassay).

  For example, to facilitate purification or detection, the LCE protein, fragment, or derivative thereof is optionally fused or chimeric protein product (ie, the LCE protein is conjugated via a peptide bond to a heterologous protein sequence of a different protein. It can be expressed as A chimeric product can be made by ligating together appropriate nucleic acid sequences encoding the desired amino acids using standard methods and expressing the chimeric product. Chimeric products can also be made by protein synthesis techniques such as the use of peptide synthesizers (Hunkapiller et al., Nature, 1984, 310: 105-111).

  Once recombinant cells expressing the LCE gene sequence have been identified, standard methods (eg, ion exchange chromatography, affinity chromatography, and gel exclusion chromatography; centrifugation; solubility differences; electrophoresis, purification references) ) Can be used to isolate and purify gene products. Alternatively, native LCE protein can be purified from natural sources by standard methods (eg immunoaffinity purification). Once the protein is obtained, it can be quantified and measured for its activity by an appropriate method such as immunoassay, bioassay, or measurement of other physical properties such as crystallography.

  In the methods of the present invention, cells engineered to change (to be misexpressed) the expression of other genes associated with the LCE or p53 pathway can also be used. As used herein, misexpression includes ectopic expression, overexpression, underexpression, and no expression (eg, due to knockout of a gene or block of expression normally caused normally).

Genetically modified animals Animals that have been genetically modified to alter LCE expression in order to test the activity of candidate p53 modulators or to further evaluate the role of LCE in the p53 pathway processes such as cell death and cell proliferation The model can be used in an in vivo assay. Preferably, altered LCE expression results in a detectable phenotype, such as reduced or increased levels of cell proliferation, angiogenesis, or cell death compared to control animals with normal LCE expression. The genetically modified animal may further have altered p53 expression (eg, p53 knockout). Preferred genetically modified animals are mammals such as primates, rodents (preferably mice), cows, horses, goats, sheep, pigs, dogs and cats. Preferred non-mammalian species include zebrafish, C. elegans, and Drosophila. Preferred genetically modified animals are described by transgenic animals, i.e. mosaic animals (e.g. Jakobovits, 1994, Curr. Biol., 4: 761-763) having heterologous nucleic acids present as extrachromosomal elements within a portion of their cells. Or a transgenic animal in which heterologous nucleic acid is stably integrated into germline DNA (ie in most or all genomic sequences of cells). Heterologous nucleic acid is introduced into the germline of such transgenic animals, for example, by genetic manipulation of embryos or embryonic stem cells of the host animal.

  Methods for producing transgenic animals are well known in the art (transgenic mice include Brinster et al., Proc. Nat. Acad. Sci. USA, 82: 4438-4442, 1985, both of which are US patents by Leder et al. US Pat. Nos. 4,736,866 and 4,870,009; US Pat. No. 4,873,191 by Wagner et al. And Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring, NY See Harbor, 1986; see Sandford et al., US Pat. No. 4,945,050 for particle bombardment; Rubin and Spradling, Science, 1982, 218: 348-53 and US Pat. See 4,670,388; for transgenic insects, see Berghammer AJ et al., A Universal Marker for Transgenic Insects, 1999 , Nature, 402: 370-371; for transgenic zebrafish, see Lin S., Transgenic Zebrafish, Methods Mol Biol., 2000, 136: 375-3830); microinjection in fish, amphibian eggs and birds See Houdebine and Chourrout, Experientia, 1991, 47: 897-905; for transgenic rats, see Hammer et al., Cell, 1990, 63: 1099-1112; cultured embryonic stem (ES) cells Then, for the production of transgenic animals by introducing DNA into ES cells using methods such as electroporation, calcium phosphate / DNA precipitation, direct injection, for example, Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, EJRobertson Volume, IRL Press, 1987). Non-human transgenic animals can be generated according to available methods (see Wilmut, I. et al., 1997, Nature, 385: 810-813; see PCT International Publication Nos. WO 97/07668 and WO 97/07669).

  In one embodiment, the transgenic animal preferably exhibits a heterozygous or homozygous change in the sequence of the endogenous LCE gene that results in reduced LCE function such that LCE expression is undetectable or negligible. Having a “knockout” animal. Knockout animals are usually produced by homologous recombination using a vector containing a transgene having at least a portion of the gene to be knocked out. Usually, in order to functionally disrupt the transgene, deletions, additions or substitutions are introduced into it. The transgene may be a human gene (eg, derived from a human genomic clone), but more preferably is an ortholog of a human gene derived from a transgenic host species. For example, the mouse LCE gene is used to construct a homologous recombination vector suitable for altering the endogenous LCE gene in the mouse genome. Detailed methods of homologous recombination in mice are available (see Capecchi, Science, 1989, 244: 1288-1292; Joyner et al., Nature, 1989, 338: 153-156) non-rodent mammals and Procedures for generating transgenics for other animals are also available (Houdebine and Chourrout, supra; Pursel et al., Science, 1989, 244: 1281-1288; Simms et al., Bio / Technology, 1988, 6: 179. ~ 183). In a preferred embodiment, a knockout animal such as a mouse in which a particular gene is knocked out can be used to produce antibodies against the human counterpart of the knocked out gene (Claesson MH et al., 1994, Scan J Immunol, 40: 257-264; Declerck PJ et al., 1995, J Biol Chem., 270: 8397-400).

  In another embodiment, the transgenic animal is an LCE gene, eg, by introducing additional copies of the LCE, or by operably inserting control sequences that alter the expression of the endogenous copy of the LCE gene. A “knock-in” animal that has changes in its genome that result in changes in expression (eg, increased expression (including increased ectopicity) and decreased). Such regulatory sequences include inducible, tissue-specific and constitutive promoter and enhancer elements. This knock-in can be homozygous or heterozygous.

  Transgenic non-human animals can also be generated that contain selected systems that allow for restricted transgene expression. An example of such a system that can be made is the cre / loxP recombinase system of bacteriophage P1 (Lakso et al., PNAS, 1992, 89: 6232-6236; US Pat. No. 4,959,317). When using the cre / loxP recombinase system to control transgene expression, an animal containing a transgene encoding both the Cre recombinase and the selected protein is required. Such animals can be transformed into “double” transgenic animals, for example, by crossing two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. It can be provided by making. Another example of a recombinase system is the Saccharomyces cerevisiae FLP recombinase system (O'Gorman et al., 1991, Science, 251: 1351-1355; US Pat. No. 5,654,182). In a preferred embodiment, both Cre-Loxp and Flp-Frt are used in the same system to control transgene expression and so that vector sequences in the same cell are sequentially deleted (Sun X Et al., 2000, Nat Genet, 25: 83-6).

  Using genetically modified animals p53 as an animal model of diseases and disorders related to defective p53 function in genetics studies and for in vivo testing of candidate therapeutics such as those identified in the screening described below The path can be further elucidated. This candidate therapeutic agent is administered to a genetically modified animal with altered LCE function, and a phenotypic change is given to a genetically modified animal that has been treated with a placebo and / or an animal in which LCE expression has not been altered to which a candidate therapeutic agent has been given. Compare to appropriate control animals.

  In addition to the above-described genetically modified animals with altered LCE function, animal models with defective p53 function (and otherwise normal LCE function) can be used in the methods of the invention. For example, p53 knockout mice can be used to assess in vivo the activity of candidate p53 modulators identified in one of the in vitro assays described below. p53 knockout mice have been described in the literature (Jacks et al., Nature, 2001; 410: 1111-1116, 1043-1044; Donehower et al., supra). Preferably, when a candidate p53 modulating agent is administered to a model system having cells that are defective in p53 function, it results in a phenotypic change detectable in the model system, thereby repairing p53 function. That is, it is shown that the cells show normal cell cycle progression.

Modulators The present invention provides methods for identifying agents that interact with and / or modulate the function of LCE and / or the p53 pathway. Such agents are useful for various diagnostic and therapeutic uses associated with the p53 pathway, and for a more detailed analysis of LCE proteins and their contribution in the p53 pathway. Accordingly, the present invention also provides a method of modulating the p53 pathway comprising the step of specifically modulating LCE activity by administering an LCE interacting or modulating agent.

  In a preferred embodiment, the LCE modulating agent is a normal LCE function, including transcription, protein expression, protein localization, cellular activity or extracellular activity, by inhibiting or enhancing LCE activity or otherwise. To affect. In a further preferred embodiment, the candidate p53 pathway modulator specifically modulates LCE function. The expressions “specific modulator”, “specifically modulate” and the like are used herein to bind directly to an LCE polypeptide or nucleic acid, preferably to inhibit, enhance or otherwise alter the function of LCE. Used to tell the regulator to make. The term also encompasses modulators that alter the interaction of the LCE with the binding partner or substrate (eg, by binding to the binding partner of the LCE or the protein / binding partner complex to inhibit function). To do.

  Preferred LCE modulators include small molecule compounds; LCE interacting proteins; and nucleic acid modulators such as antisense and RNA inhibitors. The modulator may be formulated into the pharmaceutical composition as a composition that may include other active ingredients and / or suitable carriers and excipients, such as in combination therapy. Techniques for formulating or administering compounds appear in “Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., 19th Edition.

Small molecule modulators Small molecules often have enzymatic function and / or preferably modulate the function of a protein comprising a protein interaction domain. Chemical agents referred to in the art as “small molecule” compounds are typically organic non-peptide molecules having a molecular weight of less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably less than 500. . This class of modulators includes chemically synthesized molecules such as compounds from combinatorial chemical libraries. Synthetic compounds can also be rationally designed or identified based on the properties of known or putative LCE proteins, or can be identified by screening compound libraries. Appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants and fungi that can also be identified by screening compound libraries to look for LCE modulating activity. Methods for making and obtaining compounds are well known in the art (Schreiber SL, Science, 2000, 151: 1964-1969; Radmann J and Gunther J, Science, 2000, 151: 1947-1948).

  Small molecule modulators identified from the screening assays described below can be used as lead compounds, from which candidate clinical compounds can be designed, optimized and synthesized. Such clinical compounds may be useful for treating conditions associated with the p53 pathway. The activity of candidate small molecule modulators may be improved several fold by repetitive secondary functional verification, structure determination, and candidate modulator modification and testing as described further below. In addition, candidate clinical compounds are made with particular attention to clinical and pharmacological properties. For example, reagents can be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity in pharmaceutical development.

Protein Modulators Specific LCE interacting proteins are useful in a variety of diagnostic and therapeutic applications associated with the p53 pathway and related diseases, as well as validation assays for other LCE modulating agents. In preferred embodiments, the LCE interacting protein affects normal LCE function, including transcription, protein expression, protein localization, cellular activity or extracellular activity. In another embodiment, the LCE interacting protein is useful for detecting and providing information regarding the function of the LCE protein (eg, for diagnostic means) as it is associated with p53 related diseases such as cancer. .

  LCE interacting proteins are endogenous, such as members of the LCE pathway that regulate LCE expression, localization, and / or activity, ie, those that interact genetically or biochemically naturally with LCE. Good. LCE modulators include LCE interacting proteins and dominant negative forms of the LCE protein itself. Yeast two hybrids and mutant screening provide a preferred method for identifying endogenous LCE interacting proteins (Finley, RL et al., 1996, DNA Cloning-Expression Systems: A Practical Approach, Glover D. and Hames BD Ed., Oxford University Press, Oxford, UK, pages 169-203; Fashema SF et al., Gene, 2000, 250: 1-14; Drees BL, Curr Opin Chem Biol, 1999, 3: 64-70; Vidal M and Legrain P Nucleic Acids Res, 1999, 27: 919-29; US Pat. No. 5,928,868). A preferred alternative method for elucidating protein complexes is mass spectrometry (eg, Pandley A and Mann M, Nature, 2000, 405: 837-846; Yates JR 3rd, Trends Genet, 2000, 16: 5 ~ 8 reviews).

  The LCE interacting protein may be an exogenous protein such as an antibody specific for LCE or a T cell antigen receptor (eg, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory; Harlow and Lane, 1999, Using antibodies: a laboratory manual. See Cold Spring Harbor Loboratory Press, NY). LCE antibodies are further discussed below.

  In a preferred embodiment, the LCE interacting protein specifically binds to the LCE protein. In preferred alternative embodiments, the LCE modulating agent binds to an LCE substrate, binding partner, or cofactor.

Antibodies In another embodiment, the protein modulator is an antibody agonist or antagonist specific for LCE. This antibody has therapeutic and diagnostic applications and can be used in screening assays to identify LCE modulators. The antibody can also be used in the analysis of various cellular responses and parts of the LCE pathway responsible for normal processing and maturation of LCE.

Antibodies that specifically bind to LCE polypeptides can be generated using well-known methods. Preferably, the antibody is specific for a mammalian ortholog of LCE polypeptide, more preferably human LCE. Antibodies include polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ′) ₂ fragments, fragments produced by FAb expression libraries, anti-idiotypes (anti-Id) It may be an antibody and any of the above epitope-binding fragments. For example, by routine LCE polypeptide screening to look for antigenicity against the amino acid sequence shown in any of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15 or 16, or the antigenic region of a protein against this By applying a theoretical method of selection, epitopes of LCE that are particularly antigenic can be selected (Hopp and Wood, 1981, Proc. Nati. Acad. Sci. USA, 78: 3824-28; Hopp and Wood, 1983, Mol. Immunol., 20: 483-89; Sutcliffe et al., 1983, Science, 219: 660-66). Monoclonal antibodies with affinities of 10 ⁸ M ⁻¹ , preferably 10 ⁹ M ⁻¹ to 10 ¹⁰ M ⁻¹ , or stronger can be generated by the standard procedures described (Harlow and Lane, supra). Goding, 1986, Monoclonal Antibodies: Principles and Practice (2nd edition), Academic Press, New York; US Pat. No. 4,381,292; US Pat. No. 4,451,570; US Pat. No. 4,618, 577). Antibodies against crude cell extracts of LCE or substantially purified fragments thereof can be generated. When using LCE fragments, these preferably comprise at least 10 and more preferably at least 20 consecutive amino acids of the LCE protein. In certain embodiments, LCE-specific antigens and / or immunogens are bound to carrier proteins that stimulate the immune response. For example, the polypeptide of interest is covalently bound to a keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant that enhances the immune response. Immunize an appropriate immune system such as experimental rabbits or mice according to conventional protocols.

  The presence of antibodies specific for LCE was assayed by a suitable assay, such as a solid phase enzyme-linked immunosorbent assay (ELISA) using an immobilized corresponding LCE polypeptide. Other assays such as radioimmunoassay and fluorescence assay can also be used.

  Chimeric antibodies specific to LCE polypeptides can be made that contain different portions from different animal species. For example, a human immunoglobulin constant region may be linked to a murine mAb variable region such that the biological activity of the antibody is derived from a human antibody and its binding specificity is derived from a murine fragment. Chimeric antibodies are made by splicing genes encoding appropriate regions from each species (Morrison et al., Proc. Natl. Acad. Sci., 1984, 81: 6851-6855; Neuberger et al., Nature, 1984, 312: 604-608; Takeda et al., Nature, 1985, 31: 452-454). A humanized antibody, which is a form of chimeric antibody, can be obtained by recombinant DNA technology (Riechmann LM et al., 1988, Nature, 323: 323-327). Can be made by transplanting into the background of the area (Carlos, TM, JMHarlan, 1994, Blood, 84: 2068-2101). Humanized antibodies contain about 10% murine and about 90% human sequences, which further reduces or eliminates immunogenicity while retaining antibody specificity (Co MS and Queen C., 1991). Year, Nature, 351: 501-501; Morrison SL., 1992, Ann. Rev. Immun., 10: 239-265). Humanized antibodies and methods for producing them are well known in the art (US Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370). issue).

  LCE-specific single-chain antibodies, which are recombinant single-chain polypeptides formed by linking heavy and light chain fragments of the Fv region by amino acid crosslinking, can be produced by methods well known in the art (US Patent 4,946,778; Bird, Science, 1988, 242: 423-426; Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85: 5879-5883; Ward et al., Nature, 1989. 334: 544-546).

  Other suitable techniques for producing antibodies include exposing lymphocytes in vitro to selected polypeptides libraries in antigenic polypeptides or alternatively in phage or similar vectors (Huse et al., Science, 1989). 246: 1275-1281). T cell antigen receptors as used herein are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).

  The polypeptides and antibodies of the present invention can be used with or without modification. In many cases, antibodies are labeled by either covalently or non-covalently binding a substrate that is toxic to cells that expresses a detectable signal or a target protein (Menard S et al., Int J Biol Markers, 1989, 4: 131-134). A wide variety of labels and conjugation techniques are known and widely reported in both scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorinated lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (US Pat. No. 3, 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; 4,366 241). Recombinant immunoglobulins may also be produced (US Pat. No. 4,816,567). By conjugating with a transmembrane toxin protein, an antibody against the cytoplasmic polypeptide can be delivered and reached at its target (US Pat. No. 6,086,900).

  For therapeutic use in patients, the antibodies of the invention are administered to the target site by parenteral administration or by intravenous administration when possible. Clinical studies will determine therapeutically effective doses and regimens. Usually, the amount of antibody administered is about 0.1 mg to about 10 mg per kg patient weight. For parenteral administration, the antibody is formulated in a unit dose injectable form (eg, solution, suspension, emulsion) containing a pharmaceutically acceptable vehicle. Such vehicles are essentially non-toxic and have no therapeutic effect. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as non-volatile oil, ethyl oleate, or liposomal carriers may also be used. The vehicle may contain minor amounts of additives such as buffers and preservatives that enhance isotonicity, chemical stability, or otherwise enhance the therapeutic potential. The antibody concentration in such a vehicle is usually about 1 mg / ml to about 10 mg / ml. Immunotherapeutic methods are further described in the literature (US Pat. No. 5,859,206; International Publication No. WO0073469).

Specific Biotherapy In a preferred embodiment, the LCE interacting protein is applicable to biotherapy. Biotherapeutic agents are formed into pharmaceutically acceptable carriers and can be administered to activate or inhibit signal transduction pathways. This modulation can be done by binding to the ligand and thus inhibiting the activity of the pathway, or binding to the receptor and inhibiting or activating the activity of the receptor. Alternatively, the biotherapeutic agent itself may be a ligand capable of activating or inhibiting the receptor. Biotherapeutic agents and methods for producing biotherapeutic agents are described in detail in US Pat. No. 6,146,628.

  LCE ligands, ligand antibodies, or LCE itself may be used as a biotherapeutic agent to modulate the activity of LCE in the p53 pathway.

Nucleic Acid Modulators Other preferred LCE modulators include nucleic acid molecules such as antisense oligomers and double stranded RNA (dsRNA) that generally inhibit LCE activity. Preferred nucleic acid modulators are DNA replication, transcription, translocation of LCE RNA to protein translation sites, translation of proteins from LCE RNA, splicing LCE RNA to obtain one or more mRNA species, or to LCE RNA Interfering with the function of LCE nucleic acids, such as catalytic activity that may be involved or promoted thereby

  In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to bind to LCE mRNA, preferably by binding to the 5 'untranslated region, to prevent translation. Antisense oligonucleotides specific for LCE are preferably in the range of at least 6 to about 200 nucleotides. In some embodiments, the oligonucleotide is preferably at least 10, 15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably less than 50, 40, or 30 nucleotides in length. The oligonucleotide can be single-stranded or double-stranded DNA or RNA, or a chimeric mixture or derivative thereof, or a modified version thereof. The base moiety, sugar moiety, or phosphate backbone of this oligonucleotide may be modified. The oligonucleotide may contain other accessory groups such as peptides, agents that facilitate transport across the cell membrane, hybridization-triggered cleaving agents, intercalating agents, and the like.

  In another embodiment, the antisense oligomer is a phosphothioate morpholino oligomer (PMO). PMOs are assembled from four different morpholino subunits, each containing one of the four gene bases (A, C, G, or T) attached to the six-membered ring of morpholine. Yes. The polymers of these subunits are linked by linkages between nonionic phosphodiamidate subunits. Detailed methods for making and using PMO and other antisense oligomers are well known in the art (eg, International Publication No. WO 99/18193; Probst JC, Antisense Oligodeoxynucleotide and Ribozyme Design, Methods., 2000, 22). (3): 271-281; Summerton J and Weller D., 1997, Antisense Nucleic Acid Drug Dev., 7: 187-95; US Pat. No. 5,235,033; US Pat. No. 5,378,841 reference).

  A preferred alternative LCE nucleic acid modulator is a double-stranded RNA species that mediates RNA interference (RNAi). RNAi is a sequence-specific post-translational gene silencing process in animals and plants, initiated by double-stranded RNA (dsRNA) with a sequence homologous to the gene being silenced. Methods relating to the use of RNAi to silence genes in nematodes, fruit flies, plants, and humans are well known in the art (Fire A et al., 1998, Nature, 391: 806-811; Fire, A. Trends Genet., 15, 358-363, 1999; Sharp, PA, RNA interference 2001., Genes Dev., 15, 485-490, 2001; Hammond, SM et al., Nature Rev. Genet., 2, 110 ~ 1119, 2001; Tuschl, T., Chem. Biochem., 2, 239-245, 2001; Hamilton, A. et al., Science, 286, 950-952, 1999; Hammond, SM et al., Nature, 404 293-296, 2000; Zamore, PD et al., Cell, 101, 25-33, 2000; Bernstein, E. et al., Nature, 409, 363-366, 2001; Elbashir, SM et al., Genes Dev., 15 188-200, 2001; International Publication No. WO0129058; International Publication No. WO9932619; Elbashir SM et al., 2001, Nature, 411: 494-498).

  Nucleic acid modulators are generally used as search reagents, diagnostic agents, and therapeutic agents. For example, antisense oligonucleotides that can inhibit gene expression with strict specificity are often used to elucidate the function of a particular gene (see, eg, US Pat. No. 6,165,790). . Nucleic acid modulators are also used, for example, to identify the function of various members of a biological pathway. For example, antisense oligomers have been used as a therapeutic part in the treatment of diseased animals and humans and have been demonstrated in numerous clinical trials to be safe and effective (Milligan JF et al., Current Concepts in Antisense Drug Design, J Med, Chem., 1993, 36: 1923-1937; Tonkinson JL et al., Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents, Cancer Invest., 1996, 14: 54-65). Accordingly, in one aspect of the invention, LCE specific nucleic acid modulators are used in assays to further elucidate the role of LCE in the p53 pathway and / or the relationship between LCE and other members of this pathway. In another aspect of the invention, antisense oligomers specific for LCE are used as therapeutic agents to treat conditions associated with p53.

Assay System The present invention provides an assay system and screening method for identifying specific modulators of LCE activity. As used herein, an “assay system” includes all components necessary to perform an assay that detects and / or measures a specific event and analyze the results. In general, primary assays are used to identify or confirm the specific biochemical or molecular effects of a modulator on LCE nucleic acids or proteins. In general, secondary assays may further evaluate the activity of LCE modulators identified by the primary assay and confirm that the modulator affects LCE in a manner related to the p53 pathway. In some cases, LCE modulators are tested directly in a secondary assay.

  In a preferred embodiment, the screening method comprises an LCE polypeptide under conditions that result in a control activity (eg, enzymatic activity) based on the particular molecular event detected by the screening method in the absence of the candidate agent. Contacting the assay system with a candidate agent. A statistically significant difference between the agent-affected activity and the control activity indicates that this candidate agent modulates LCE activity and thus the p53 system.

Primary Assay Generally, the type of primary assay is determined by the type of modulator being tested.

Primary assays for small molecule modulators For small molecule modulators, screening assays are used to identify candidate modulators. Screening assays may be cell based and may use a cell-free system that reinitiates or retains the biochemical response associated with this target protein (Sittampalam GS et al., Curr Opin Chem Biol, 1997). 1: 384-91 and accompanying references are reviewed). As used herein, the term “cell-based” refers to assays using live cells, dead cells, or specific cell fractions such as membrane fraction, endoplasmic reticulum fraction, mitochondrial fraction, and the like. The term “cell-free” encompasses assays using substantially purified proteins (endogenous or recombinantly produced), partially purified or crude cell extracts. Screening assays include protein-DNA interactions, protein-protein interactions (eg, receptor-ligand binding), transcriptional activity (eg, reporter genes), enzyme activity (eg, via substrate characteristics), second messenger activity, immunity Various molecular events can be detected, including protogenicity, and changes in cell morphology and other cellular characteristics. Appropriate screening assays can use a wide range of detection methods, including fluorescence, radioactivity, colorimetry, spectrophotometry, and amperometry, to provide a readout of the specific molecular events to detect.

Cell-based screening assays typically require a system that recombinantly expresses LCE and any auxiliary proteins required by the individual assay. Appropriate methods for generating recombinant proteins retain sufficient biological activity and produce sufficient amounts of protein sufficient to optimize the activity and ensure assay reproducibility. Yeast two-hybrid screening, mutant screening, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidating protein complexes. For certain applications, when using LCE-interacting proteins for screening to identify small molecule modulators, the binding specificity of the interacting protein for LCE protein can be determined by treatment with a substrate (eg, an agent that specifically binds to a candidate LCE). The ability to function as a negative effector in LCE-expressing cells), binding equilibrium constants (usually at least about 10 ⁷ M ⁻¹ , preferably at least about 10 ⁸ M ⁻¹ , more preferably at least about 10 ⁹ M ⁻¹ ), It can be assayed by a variety of well-known methods such as immunogenicity (eg, the ability to elicit antibodies specific for LCE in heterologous hosts such as mice, rats, goats or rabbits). For enzymes and receptors, binding can be assayed by treatment with substrate and ligand, respectively.

  In screening assays, the ability of a candidate agent to specifically bind to or modulate the activity of an LCE polypeptide, a fusion protein thereof, or a cell or membrane containing the polypeptide or fusion protein can be measured. The LCE polypeptide can be full length or a fragment thereof that retains functional LCE activity. The LCE polypeptide may be fused to another polypeptide, such as a peptide tag for detection or immobilization or another tag. The LCE polypeptide is preferably human LCE, or an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects a modulation based on a candidate agent for interaction of the LCE with a binding target, such as an endogenous protein, exogenous protein, or other substrate having specific binding activity for LCE, This can be used to assess normal LCE gene function.

  Appropriate assay formats that can be adapted for screening to look for LCE modulators are well known in the art. Preferred screening assays are high-throughput or ultra-high throughput, thus providing an automated and cost-effective means of screening compound libraries for lead compounds (Fernandes PB, Curr Opin Chem Biol, 1998, 2: 597-603; Sundberg SA, Curr Opin Biotechnol, 2000, 11: 47-53). In a preferred embodiment, the screening assay uses fluorescence techniques including fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer. These systems provide a means to monitor protein-protein or DNA-protein interactions where the intensity of the signal emitted from the dye-labeled molecule depends on its interaction with its partner molecule (eg, Selvin PR Nat Struct Biol, 2000, 7: 730-4; Fernandes PB, supra; Hertzberg RP and Pope AJ, Curr Opin Chem Biol, 2000, 4: 445-451).

  A variety of suitable assay systems can be used to identify candidate LCE and p53 pathway modulators (eg, US Pat. No. 6,020,135 (modulation of p53). Preferred specific assays are detailed below. Describe.

Elongase assay. Extension enzyme assays are well known in the art. In one example of an extension assay, chromatographic (eg, HPLC) analysis of the labeled extension product is performed to assess LCE activity. Long chain fatty acids can be labeled with 14C (Moon YA et al., 2001, J Biol Chem 276: 45358-45366). Briefly, fatty acid elongation activity is measured in microsomes prepared from transfected cells. Fatty acyl-CoA or BSA conjugated fatty acids can be used as substrates for the reaction. The substrate mixture also contains [2- ¹⁴ C] malonyl-CoA. The elongation reaction is initiated when the microsomal protein and the substrate are mixed. After stopping the reaction, the fatty acid is recovered, washed and counted. Elongation enzyme activity is measured as the amount of radioactivity introduced into the fatty acid and is expressed as that amount.

  Apoptosis assay. The cell death assay can be performed by a terminal deoxynucleotidyl transferase mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay. The TUNEL assay measures nuclear DNA fragmentation characteristic of cell death by following fluorescein-dUTP incorporation (Yonehara et al., 1989, J. Exp. Med., 169, 1747) (Lazebnik et al., 1994). , Nature, 371, 346). Cell death can be further assayed by acridine orange staining of tissue culture cells (Lucas, R. et al., 1998, Blood, 15: 4730-41). Apoptosis assay systems can include cells that express LCE, and optionally those that have defective p53 function (eg, p53 is overexpressed or underexpressed compared to wild type cells). A test agent can be added to the apoptosis assay system, and a change in induction of cell death compared to a control without the test agent identifies a candidate p53 modulating agent. In some embodiments of the invention, an apoptosis assay can be used as a secondary assay to test candidate p53 modulators initially identified using a cell-free system. Apoptosis assays can also be used to test whether LCE function plays a direct role in cell death. For example, an apoptosis assay can be performed on cells that over- or under-express LCE compared to wild-type cells. Differences in cell death response compared to wild type cells suggests that LCE plays a direct role in the cell death response. Apoptosis assays are further described in US Pat. No. 6,133,437.

  Cell proliferation and cell cycle assays. Cell proliferation can be assayed via bromodeoxyuridine (BRDU) incorporation. In this assay, BRDU is incorporated into newly synthesized DNA to identify the cell population in which the DNA is synthesized. Subsequently, using anti-BRDU antibodies (Hoshino et al., 1986, Int. J. Cancer, 38, 369; Campana et al., 1988, J. Immunol. Meth., 107, 79) or by other means, Synthesized DNA can be detected.

[ ³ H] -thymidine incorporation can also be used to examine cell proliferation (Chen, J., 1996, Oncogene, 13: 1395-403; Jeoung, J., 1995, J. Biol Chem., 270: 18367-73). This assay allows quantitative characterization of S-phase DNA synthesis. In this assay, cells synthesizing DNA incorporate [ ³ H] -thymidine into newly synthesized DNA. Uptake can then be measured by standard techniques such as radioisotope counting with a scintillation counter (eg, a Beckman LS 3800 liquid scintillation counter).

  Cell proliferation can also be assayed by colony formation in soft agar (Sambrook et al., Molecular Cloning, Cold Spring Harbor, 1989). For example, cells transformed with LCE are seeded on soft agar plates, incubated for 2 weeks, and then colonies are measured and counted.

  Flow cytometry can assay gene involvement in the cell cycle (Gray JW et al., 1986, Int J Radiat Biol Relat Stud Phys Chem Med, 49: 237-55). Cells transfected with LCE can be stained with propidium iodide and evaluated by flow cytometry (available from Becton Dickinson).

  Thus, cell proliferation or cell cycle assay systems include cells that express LCE and optionally those that have defective p53 function (eg, p53 is overexpressed or underexpressed compared to wild type cells). Can do. A test agent can be added to the assay system, and changes in cell proliferation or cell cycle relative to controls where no test agent is added, identify candidate p53 modulating agents. In some embodiments of the invention, a cell proliferation or cell cycle assay is used as a secondary assay to test candidate p53 modulators initially identified using another assay system, such as a cell-free kinase assay system. be able to. Cell proliferation assays can also be used to test whether LCE function plays a direct role in cell proliferation or cell cycle. For example, cell proliferation or cell cycle assays can be performed on cells that over- or under-express LCE compared to wild-type cells. Differences in proliferation or cell cycle compared to wild type cells suggests that LCE plays a direct role in cell proliferation or cell cycle.

  Angiogenesis. Various human endothelial cell lines such as umbilical cord, coronary artery, or dermal cells can be used to assay angiogenesis. Suitable assays include an Alamar Blue based assay that measures proliferation (available from Biosource International); a Becton Dickinson Falcon that measures migration of cells through the membrane in the presence or absence of an angiogenic enhancer or suppressor Migration assays using fluorescent molecules such as the use of HTS FluoroBlock cell culture inserts; tubule formation assays based on the formation of tubular structures by endothelial cells on Matrigel® (Becton Dickinson). Thus, angiogenesis assay systems can include cells that express LCE, and optionally those that have defective p53 function (eg, p53 is overexpressed or underexpressed compared to wild type cells). Test agents can be added to the angiogenesis assay system, and changes in angiogenesis compared to controls where no test agent is added, identify candidate p53 modulating agents. In some embodiments of the invention, an angiogenesis assay can be used as a secondary assay to test candidate p53 modulators initially identified using another assay system. An angiogenesis assay can also be used to test whether LCE function plays a direct role in cell proliferation. For example, an angiogenesis assay can be performed on cells that over- or under-express LCE compared to wild-type cells. Differences in angiogenesis compared to wild type cells suggests that LCE plays a direct role in angiogenesis.

  Hypoxia induction. The alpha subunit of hypoxia-inducible factor-1 (HIF-1), a transcription factor, is upregulated in tumor cells after exposure to hypoxia in vitro. Under hypoxic conditions, HIF-1 stimulates the expression of genes known to be important for tumor cell survival, such as glycolytic enzymes and genes encoding VEGF. Induction of such genes by hypoxic conditions uses cells transfected with LCE (eg, 0.1% O 2, 5% CO 2 generated in a Napco 7001 incubator (Precision Scientific), and the rest N 2 Can be assayed by growing under hypoxic and normoxic conditions and then assessing the activity or expression of the gene by Taqman®. For example, hypoxic induction assay systems can include cells that express LCE and optionally have mutated p53 (eg, p53 is overexpressed or underexpressed compared to wild type cells). . Test agents can be added to the hypoxic induction assay system, and changes in hypoxic response relative to controls where no test agent is added, identify candidate p53 modulating agents. In some embodiments of the invention, the hypoxic induction assay can be used as a secondary assay to test candidate p53 modulators initially identified using another assay system. A hypoxic induction assay can also be used to test whether LCE function plays a direct role in the hypoxic response. For example, a hypoxic induction assay can be performed on cells that over- or under-express LCE compared to wild-type cells. Differences in hypoxic response compared to wild type cells suggests that LCE plays a direct role in hypoxic induction.

  Cell adhesion. In cell adhesion assays, the adhesion between cells and purified adhesion protein or the mutual adhesion of cells in the presence or absence of a candidate modulator is measured. In a cell-protein adhesion assay, the ability of the agent to modulate cell adhesion to purified protein is measured. For example, recombinant protein is produced, diluted to 2.5 g / mL with PBS and used to coat the wells of a microtiter plate. Do not coat wells used as negative controls. The coated wells are then washed, blocked with 1% BSA, and washed again. Compounds are diluted to 2 × final test concentration and added to the blocked, coated wells. Thereafter, cells are added to the wells and unbound cells are washed away. The retained cells are labeled directly on the plate by adding a membrane permeable fluorescent dye such as calcein-AM and the signal is quantified with a fluorescent microplate reader.

  In a cell-cell adhesion assay, the ability of an agent to modulate cell adhesion protein binding to a native ligand is measured. These assays use cells that express adhesion proteins selected either naturally or recombinantly. In an exemplary assay, cells expressing cell adhesion proteins are seeded into the wells of a multiwell plate. Cells expressing the ligand are labeled with a membrane permeable fluorescent dye such as BCECF and allowed to adhere to the monolayer in the presence of the candidate agent. Unbound cells are washed away and bound cells are detected using a fluorescence plate reader.

  A high throughput cell adhesion assay has also been described. In one such assay, a microarray spotter is used to bind small molecule ligands and peptides to the surface of the microscope slide, after which untreated cells are contacted with the slide and unbound cells are washed away. In this assay, not only the binding specificity of peptides and modulators to cell lines is determined, but also functional cell signaling of attached cells is measured in situ using immunofluorescence technology on a microchip. (Falsey JR et al., Bioconjug Chem., May-June 2001, 12 (3): 346-53).

  Tubule formation. Tubulogenesis assays monitor the ability of cultured cells, usually endothelial cells, to form tubular structures on a matrix substrate that typically stimulates the environment of the extracellular matrix. Exemplary substrates include Matrigel® (Becton Dickinson), an extract of a basement membrane protein containing laminin, and collagen IV, and a heparin sulfate proteoglycan that is liquid at 4 ° C. and becomes a hard gel at 37 ° C. included. Other suitable substrates include collagen, fibronectin and / or fibrin. Cells are stimulated with an angiogenesis stimulating agent, and their ability to form tubules is detected by imaging. Usually, tubules are detected after overnight incubation with stimuli, but longer or shorter times may be employed. Tube formation assays are also well known in the art (eg, Jones MK et al., 1999, Nature Medicine 5: 1418-1423). These assays traditionally use stimulation with serum or stimulation with growth factors FGF or VEGF. Serum means a non-limiting source of growth factor. In a preferred embodiment, cells cultured in a serum free medium are assayed to control the process or pathway by which the candidate drug is modulated. Furthermore, it has been discovered that different target genes have another response to stimulation by different pro-angiogenic agents including inflammatory angiogenic factors such as TNF alpha. Thus, in a further preferred embodiment, the tubule formation assay system comprises testing the response of LCE to various factors such as FGF, VEGF, phorbol myristate acetate (PMA), TNF alpha, ephrin, etc. .

  Cell migration. Invasion / migration assays (also called migration assays) test the ability of cells to overcome a physical barrier and migrate towards a pro-angiogenic signal. Migration assays are well known in the art (eg, Paik JH et al., 2001, J Biol Chem. 276: 11830-11837). In a typical experimental setup, cultured endothelial cells are embedded in a thin film coated with a substrate having pores smaller in diameter than normal cells. As mentioned above, the matrix usually stimulates the environment of the extracellular matrix. The thin film is typically a membrane such as a transwell polycarbonate membrane (Corning Costar Corporation, Cambridge, Mass.) And is usually part of a lower chamber containing a pro-angiogenic stimulant and an upper chamber with a liquid contact. . Typically, overnight incubation with stimuli is followed by assaying for migration, although longer or shorter times may be employed. Assessment of migration is based on the number of cells that have passed through the membrane and can be detected using cells stained with a hemotoxylin solution (VWR Scientific, South San Francisco, Calif.) Or any other option to determine the number of cells It can be detected by the method. In another exemplary setting, cells are fluorescently labeled and migration is detected using a fluorescence reading such as Falcon HTS FluoroBlok (Becton Dickinson). Although there are movements that can be observed without stimulants, the movement is greatly increased in response to angiogenesis-promoting factors. As noted above, preferred assay systems for migration / invasion include testing the response of LCE to various pro-angiogenic factors including tumor angiogenesis and inflammatory angiogenic agents, and cell culture in serum-free media. .

  Budding assay. The budding assay is a three-dimensional in vitro angiogenesis assay that uses endothelial cell count spheroid aggregation (“spheroids”) embedded in a gel-based collagen matrix. Spheroids serve as a starting point for the budding of capillaries by entry into the extracellular matrix (referred to as “cell budding”) and subsequent complex network formation (Korff and Augustin, 1999, J Cell Sci 112: 3249-58). In one exemplary experimental setup, spheroids are prepared by pipetting 400 human umbilical vein endothelial cells per well of a non-adherent 96-well plate and performing overnight spheroid aggregation (Korff and Augustin: J Cell Biol 143: 1341-52, 1998). The spheroids are collected and spread on 900 μl of methocel collagen solution, pipetted into the core wells of a 24-well plate and allowed to polymerize the collagen gel. After 30 minutes, the reagent is added by pipetting 100 μl of a 10-fold concentrated working dilution of the test substance on top of the gel. Incubate the plate at 37 ° C. for 24 hours. The dishes are fixed at the end of the experimental incubation period by adding paraformaldehyde. By measuring the cumulative germination length per spheroid with an automated image analysis system, the germination intensity of the endothelial cells can be quantified.

Primary assays for antibody modulators For antibody modulators, a suitable primary assay test is a binding assay that tests the affinity and specificity of the antibody for LCE protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999, supra). A preferred method for detecting antibodies specific for LCE is the enzyme linked immunosorbent assay (ELISA). Other methods include FACS assays, radioimmunoassays, and fluorescence assays.

Primary assays for nucleic acid modulators For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit or enhance LCE gene expression, preferably mRNA expression. In general, expression analysis involves comparing LCE expression in similar populations of cells in the presence or absence of nucleic acid modulators (eg, two cell pools that express LCE endogenously or recombinantly). Is included. Methods for analyzing mRNA and protein expression are well known in the art. For example, LCE in cells treated with nucleic acid modulators using Northern blotting, slot blotting, RNase protection, quantitative RT-PCR (eg, using TaqMan®, PE Applied Biosystems), or microarray analysis. It can be seen that mRNA expression is reduced (eg, Current Protocols in Molecular Biology, 1994, Ausubel FM et al., John Wiley & Sons, Inc., Chapter 4; Freeman WM et al., Biotechniques, 1999 26: 112-125; Kallioniemi OP, Ann Med, 2001, 33: 142-147; Blohm DH and Guiseppi-Elie, A Curr Opin Biotechnol, 2001, 12: 41-47). Protein expression can also be monitored. Proteins are most commonly detected using specific antibodies or antisera to either LCE proteins or specific peptides. Various means are available including Western blotting, ELISA, in situ detection (Harlow E and Lane D, 1988 and 1999, supra).

Secondary assay To further assess the activity of LCE modulators identified by any of the methods described above using a secondary assay to confirm that the modulator affects LCE in a manner associated with the p53 pathway. Can do. As used herein, LCE modulators include candidate clinical compounds or other agents derived from previously identified modulators. Secondary assays can also be used to test the activity of a modulator in a particular genetic or biochemical pathway, or to test the specificity with which a modulator interacts with LCE.

  Secondary assays generally compare similar populations of cells or animals (eg, two cell pools that express LCE endogenously or recombinantly) in the presence or absence of candidate modulators. In general, in such assays, treating a cell or animal with a candidate LCE modulating agent results in a change in the p53 pathway compared to an untreated (or mock or placebo-treated) cell or animal. Test to see if it does. In certain assays, a “sensitized genetic background” is used. As used herein, “sensitized genetic background” refers to cells or animals that have been engineered to alter the expression of genes in p53 or interacting pathways.

Cell-Based Assays Cell-based assays include various mammalian cell lines known to have defective p53 function (eg, SAOS available from, among others, American Type Culture Collection (ATCC), Manassas, Va.). -2 osteoblasts, H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT-29 and DLD-1 colon cancer cells) may be used. Cell-based assays detect endogenous p53 pathway activity or may rely on recombinant expression of p53 pathway components. Any of the foregoing assays can be used in this cell-based format. Candidate modulators are usually added to the cell culture medium, but may be injected into the cells or delivered by any other effective means.

Animal Assays To test candidate LCE modulators, various non-human animal models of the normal or defective p53 pathway can be used. Usually, models of defective p53 pathways use genetically modified animals that are engineered to misexpress (eg, overexpress or lack of expression) genes involved in the p53 pathway. In general, assays require that candidate modulators be delivered systemically by oral administration, infusion, and the like.

  In a preferred embodiment, the activity of the p53 pathway is assessed by monitoring neovascularization and angiogenesis. To test the effect of candidate modulators on LCE in the Matrigel® assay, an animal model with defective and normal p53 is used. Matrigel® is an extract of basement membrane protein and is composed mainly of laminin, collagen IV, and heparin sulfate proteoglycan. This is provided as a sterile liquid at 4 ° C, but forms a gel quickly at 37 ° C. Liquid Matrigel® is mixed with various angiogenic agents such as bFGF and VEGF, or human tumor cells overexpressing LCE. This mixture is then injected subcutaneously (SC) into female athymic nude mice (Taconic, Germantown, NY) to support a vigorous vascular response. Mice with Matrigel® pellets may be dosed with the candidate modulator by the oral (PO), intraperitoneal (IP), or intravenous (IV) route. 5-12 days after injection, mice are euthanized and Matrigel® pellets are collected for hemoglobin analysis (Sigma plasma hemoglobin kit). It was found that the hemoglobin content of the gel correlates with the degree of neovascularization in the gel.

In another preferred embodiment, the effect of a candidate modulator on LCE is assessed by a tumorigenesis assay. In one example, xenograft human tumors are transplanted in SCs into 6-7 week old female athymic mice as single cell suspensions, either from existing tumors or from in vitro cultures. Tumors that endogenously express LCE are injected into the flank with a 27-gauge needle in a volume of 100 μL, 1 × 10 ⁵ to 1 × 10 ⁷ cells per mouse. Thereafter, a tag was placed on the ear of the mouse, and the tumor was measured twice a week. Treatment with the candidate modulator was started on the day when the average tumor weight reached 100 mg. Candidate modulators are delivered IV, SC, IP, or PO by bolus administration. Depending on the pharmacokinetics of each unique candidate modulator, multiple doses can be administered per day. Tumor weight was evaluated by measuring the vertical diameter with a caliper and calculated by multiplying the two dimensional diameter measurements. At the end of the experiment, the resected tumor can be used to identify biomarkers for further analysis. For immunohistochemical staining, xenograft tumors were fixed with 4% paraformaldehyde, 0.1 M phosphate, pH 7.2 for 6 hours at 4 ° C., immersed in 30% sucrose in PBS, and cooled with liquid nitrogen. Freeze quickly in isopentane.

Diagnostic and therapeutic uses Specific LCE modulators are useful in a variety of diagnostic and therapeutic applications where the disease or disease prognosis is associated with defects in the p53 pathway, such as angiogenesis, cell death, or proliferative diseases. Accordingly, the present invention provides a method of modulating the p53 pathway in a cell, preferably a cell that has been previously determined to have defective p53 function, comprising administering to the cell an agent that specifically modulates LCE activity. Also provide. Preferably, the modulator causes a detectable phenotypic change in the cell, thereby indicating that p53 function has been repaired, i.e., the cell undergoes the cell cycle with normal growth or progression, for example. .

  With the discovery that LCE is associated with the p53 pathway, various methods that can be used for diagnosis and prognostic assessment of diseases and disorders associated with defects in the p53 pathway, and identification of subjects predisposed to such diseases and disorders Is provided.

  A variety of expression analysis methods such as Northern blotting, slot blotting, RNase protection, quantitative RT-PCR, and microarray analysis can be used to diagnose whether LCE is expressed in a particular sample ( For example, Current Protocols in Molecular Biology, 1994, Ausubel FM et al., John Wiley & Sons, Inc., Chapter 4; Freeman WM et al., Biotechniques, 1999, 26: 112-125; Kallioniemi OP, Ann Med, 2001, 33: 142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol, 2001, 12: 41-47). Tissues with diseases or disorders associated with defective p53 signaling that express LCE have been identified as amenable to treatment with LCE modulating agents. In preferred applications, p53-deficient tissue overexpresses LCE compared to normal tissue. For example, Northern blot analysis of mRNA from tumors and normal cell lines, or from tumors and corresponding normal tissue samples from the same patient, using full or partial LCE cDNA sequences as probes, specific tumors expressed LCE Alternatively, it can be determined whether it is overexpressed. Alternatively, TaqMan® is used (PE Applied Biosystems) for quantitative RT-PCR analysis of LCE expression in cell lines, normal tissues and tumor samples.

  Utilizing reagents such as LCE oligonucleotides and antibodies to LCE, either (1) detection of the presence of an LCE gene mutation described above, or either overexpression or underexpression of LCE mRNA compared to a non-disease state Various for detection of (2) detection of either excess or under-existence of LCE gene products compared to non-disease states, and (3) detection of perturbations or abnormalities in LCE-mediated signaling pathways Other diagnostic methods can be implemented.

  Thus, in certain embodiments, the invention controls (a) obtaining a biological sample from a patient, (b) contacting the sample with a probe for LCE expression, (c) controlling the results from step (b). And (d) determining whether or not step (c) indicates a possible disease, is directed to a method of diagnosing a patient's disease. Preferably the disease is cancer, most preferably the cancer shown in Table 1. The probe may be either DNA or a protein including an antibody.

  The following experimental sections and examples are provided for purposes of illustration and not limitation.

I. Screening of Drosophila p53 The Drosophila p53 gene was specifically overexpressed in sputum using a trace margin margin quadrant enhancer. Increasing the amount of p53 in Drosophila (measuring titers using 1 or 2 copies of transgenic inserts of varying strength) destroys the pattern and polarity of the eyelashes, shortens and thickens the veins Moderate wrinkle deterioration with moderate phenotype, including the appearance of dark wrinkles on the wing blades, the appearance of dark “death” inclusions It was. In a screen designed to identify Drosophila p53 enhancers and suppressors, homozygous females carrying two copies of p53 were bred with 5633 males containing random inserts of piggyBac transposon (Fraser M et al., Virology, 1985, 145: 356-361). For enhancement or suppression of the p53 phenotype, progeny containing the insert were compared to sibling progeny without the insert. The modifier gene was identified using the sequence information surrounding the piggyBac insertion site. Anther phenotype modifiers have been identified as members of the p53 pathway. Drosophila LCE was an enhancer of the frog phenotype. This modifier ortholog in humans is referred to herein as LCE.

  A BLAST analysis (Altschul et al., Supra) was performed to identify targets for Drosophila modifiers. For example, the representative sequences GI # 104444345, 13129088, 12232379, and 17454617 from LCE (SEQ ID NOs: 9, 11, 14, and 16 respectively) are the Drosophila Borde spot and 45%, 48%, 25%, and Shares 41% amino acid identity.

  The various domains, signals, and functional subunits of the protein are represented by PSORT (Nakai K. and Horton P., Trends Biochem Sci, 1999, 24: 34-6; Kenta Nakai, Protein sorting signals and prediction of subcellular localization, Adv. Protein Chem. 54, 277-344 (2000), PFAM (Bateman A. et al., Nucleic Acids Res, 1999, 27: 260-2; http: //pfam.wustl.due), SMART (Ponting CP SMART: identification and annotation of domains from signaling and extracellular protein sequences. Nucleic Acids Res., January 1999 1; 27 (1): 229-32), TM-HMM (Erik LL Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences.In Proc. Of Sixth Int. Conf. On Intelligent Systems for Molecular Biology, P175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop , D. Sankoff, and C. Sensen Menlo Park, CA: AAAI Press, 1998), and crust (clus t) (Remm M and Sonnhammer E. Classification of transmembrane protein families in the Caenorhabditis elegans genome and identification of human orthologs. Genome Res. November 2000; 10 (11): 1679-89). When PFAM is used, the GCE1 / SUR4 domain (PFAM01151) of LCE of GI # 104444345, GI # 13129088, GI # 122232379, GI # 17445417 (SEQ ID NOs: 9, 11, 14 and 16 respectively) Located in groups 1-235, 10-265, 9-289, and 55-270. Furthermore, when using TM-HMM, GI # 104444345 (SEQ ID NO: 9) has seven transmembrane domains, and the amino acid positions of the start and end points are (4, 21) (26, 48) (52 , 74) (81, 103) (131, 153) (166, 188) (203, 225), and GI # 13129088 (SEQ ID NO: 11) has six transmembrane domains, and its start and end points The positions are (34, 51) (66, 88) (137, 156) (161, 183) (195, 217) (232, 254), and GI # 122232379 (SEQ ID NO: 14) has seven transmembrane domains. The start point and end point positions are (47, 64) (77, 99) (125, 147) (154, 173) (183, 205) (217, 239) (249, 267). , GI # 17445417 (allocation No. 16) has seven transmembrane domains, the start and end positions of which are (33,55) (60,82) (86,108) (115,137) (165,187) (200, 222) (237, 259).

II. High Throughput In Vitro Fluorescence Polarization Assay Fluorescently labeled LCE peptide / substrate is transferred to a 96-well microtiter plate with test agent in test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6). Added to each well. Changes in the fluorescence polarization relative to the control value determined using Fluorolite FPM-2 Fluorescence Polarization Microsystem System (Dynatech Laboratories, Inc) indicate that the test compound is a candidate modifier for LCE activity.

III. High-throughput In Vitro binding assay
³³ P-labeled LCE peptide was added to assay buffer (100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl ₂ , 1% glycerol, 0.5% NP-40, 50 mM β-mercapto together with the test agent. (Ethanol, 1 mg / ml BSA, protease inhibitor reaction mixture) was added to Neutralite-avidin coated assay plates and incubated at 25 ° C. for 1 hour. Subsequently, biotin-labeled substrate was added to each well and incubated for 1 hour. The reaction was stopped by washing with PBS and counted with a scintillation counter. Test agents that cause a change in activity relative to controls without test agent were identified as candidate p53 modulators.

IV. Immunoprecipitation and immunoblotting For co-precipitation of transfected proteins, 3 × 10 ⁶ appropriate recombinant cells containing LCE protein are planted in 10 cm dishes and transfected the next day using expression constructs. It was. The total DNA amount was kept constant at each transfection by adding empty vector. After 24 hours, cells were harvested, washed once with phosphate buffered saline, 50 mM Hepes, pH 7.9, 250 mM NaCl, 20 mM glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitro. Dissolved for 20 minutes on ice in 1 ml of lysis buffer containing phenyl phosphate, 2 mM dithiothreitol, protease inhibitors (complete, Roche Molecular Biochemicals), and 1% nonidet P-40. Cell debris was removed by two centrifugations at 15,000 xg for 15 minutes. Cell lysates were incubated with 25 μl of M2 beads (Sigma) for 2 hours at 4 ° C. with gentle rocking.

  After washing well with lysis buffer, the protein bound to the beads was dissolved by boiling in SDS sample buffer, fractionated by SDS polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane and labeled Blotted with antibody. Reactive bands were visualized by horseradish peroxidase conjugated to the appropriate secondary antibody and sensitive chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech).

V. Expression Analysis All cell lines used in the following experiments are NCI (National Cancer Center) lines and are available from ATCC (American Type Culture Collection, Manassas, VA, 2011-10209). Normal and tumor tissues were obtained from Impath, UC Davis, Clontech, Stratagene, and Ambion.

  TaqMan analysis was used to assess the expression levels of the disclosed genes in various samples.

  RNA was extracted from each tissue sample using Qiagen (Valencia, Calif.) RNeasy kit according to the manufacturer's protocol to a final concentration of 50 ng / μl. The RNA samples are then reverse transcribed using random hexamers and 500 ng total RNA of each reaction according to Protocol 4304965 of Applied Biosystems (Foster City, CA, http://www.appliedbiosystems.com/). Single stranded cDNA was synthesized.

  Primers for expression analysis using the TaqMan assay (Applied Biosystems, Foster City, CA) were prepared according to the TaqMan protocol as well as the following criteria. The criteria are a) design primer pairs across introns to eliminate genomic contamination, and b) each primer pair produces only one product.

  The Taqman reaction was performed using 300 nM primer and 250 nM probe and approximately 25 ng cDNA in a total volume of 25 μl in a 96 well plate and 10 μl total volume in a 384 well plate according to the manufacturer's protocol. A standard curve for results analysis was generated using a universal pool of human cDNA samples, which is a mixture containing cDNA from a wide range of tissues so that the target is likely to be present in large quantities. The raw data was normalized using 18S rRNA (universally expressed in all tissues and cells).

  For each expression analysis, tumor tissue samples were compared with corresponding normal tissues from the same patient. A gene is considered overexpressed in a tumor if the level of gene expression in the tumor is more than twice as high as the corresponding normal sample. If normal tissue is not available, a universal pool of cDNA samples is used instead. In these cases, the difference in expression level from the average of all normal samples from the same tissue type as the tumor sample is the standard deviation of all normal samples (ie, tumor-average (all normal samples)> 2 × STDEV A gene is considered over-expressed in a tumor sample if more than twice (all samples)).

  The results are shown in Table 1. Data in bold indicates that more than 50% of the tested tumor samples of the tissue type shown in the first column overexpressed the genes listed in column 1 compared to normal samples. Underlined data indicates that 25% to 49% of the tumor samples tested showed overexpression. By administering to a tumor in which the gene is overexpressed, the therapeutic effect of the modulator identified by the assays described herein can be further verified. Reduced tumor growth confirms the therapeutic utility of the modulator. By obtaining a tumor sample from a patient and assaying the expression of the gene targeted by the modulator, one can diagnose the likelihood that the patient will respond to treatment before treating the patient with the modulator. The expression data of this gene (or genes) can also be used as a marker for diagnosing disease progression. This assay can be performed by expression analysis as described above, by antibodies to gene targets, or by any other available detection method.

VI. The functional deficiency of the Drosophila CIG30 / LCE gene is associated with tracheal defects.
A genetic screen was designed to identify modifiers of Drosophila branching morphogenesis. Briefly, Drosophila embryos that are homozygous for the lethal insertion of piggyBac or P-element transposon (Fraser M et al., Virology, 1985, 145: 356-361) are treated with monoclonal antibodies. Screened using 2A12 (Samakovlis C, et al., Development, 1996, 122: 1395-1407; Patel NH., 1994, Practical Uses in Cell and Molecular Biology. Eds LSB Goldstein and EA Fryberg. Vol 44 pp446- 448. San diego Academic Press). Sequence information surrounding the transposon insertion site was used to identify genes mutated by insertion. A homozygous disruption of the Drosophila CIG30 (baldspot) gene was identified in association with tracheal defects.

VII. Functional deficits in zebrafish LCE are related to branching morphogenesis defects.
Antisense technology was used to identify vascular defects related to the functional deficiency of the zebrafish LCE (nucleic acid and polypeptide sequences are present in SEQ ID NOs: 7 and 15, respectively) as shown by DrCIG30L4. Ten candidate zebrafish LCE genes were identified. Four of these were tested for their involvement in angiogenesis, primarily using the following method. Wild-type single cell embryos derived from the Tubingen line were treated with antisense morpholino oligonucleotides (PMO) targeting the predicted zebrafish gene. PMO was dissolved in injection buffer (0.4 mM MgSO ₄ , 0.6 mM CaCl ₂ , 0.7 mM KCl, 58 mM NaCl, 25 mM Hepes [pH 7.6]) and 2-8 ng was dissolved in single cell stage zebra. Fish embryos were injected.

On day 4 after fertilization, larvae were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 minutes. The fixed larvae were dehydrated in methanol and stored overnight at -20 ° C. After permeabilization in acetone (−20 ° C. for 30 minutes), the embryos were washed with PBS and stained buffer (100 mM TriHCl [pH 9.5], 50 mM MgCl ₂ , 100 mM NaCl, 0.1 % Tween20) for 45 minutes. Staining buffer (stock solution: 75 mg / ml NBT in 70% N, N-dimethylformamide, 50 mg / ml BCIP in N, N-dimethylformamide) 2.25 μl nitroblue tetrazolium (NBT, Sigma) and 1. The staining reaction was started by adding 75 μl of 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma).

  Fixed specimens were scanned for modulation of angiogenesis. Treatment of embryos with PMO corresponding to the complementary strand of nucleotides 692-712 of SEQ ID NO: 7 (nucleotide sequence) resulted in angiogenesis and angiogenesis blocking depending on dose. After injecting 2 ng of PMO, the segmental vessel (ISV) was split; when the PMO was 4 ng, the ISV deletion was much more severe. After injecting 8 ng of PMO, the vascular loss was even more severe, almost free of ISV, and the intestinal vein (SIV) was almost completely destroyed.

VIII. ELOVL4 is an angiogenic gene.
Based on the analysis of zebrafish DrCIG30L4, ELOVL4 was identified as an angiogenic gene. Computer analysis, in particular BLAST, Smith-Waterman, and CLUSTALW analysis, was used to identify human ELOVL4 ("HsELOVL4", GI # 12020443 and GI # 122232379, SEQ ID NOs: 13 and 14) as an ortholog of DrCIG30L4. The Drosophila CIG30 protein sequence was BLASTed against the homosapiens amino acid sequence database to identify a family of CIG30 and ELOV (fatty acid elongase). Similarly, homosapiens sequences were used for BLAST analysis of zebrafish amino acid sequences available from both public and internal sequence databases. First, four zebrafish homologs of the CIG30 / ELOV family were identified. In order to re-evaluate the orthologs of DrCIG30L4 and other zebrafish orthologs, they were each subjected to BLAST analysis against the homosapiens amino acid sequence database. In the case of DrCIG30L4, the most “matched” was Homo sapiens ELOVL4. The homosapiens ELOVL4 amino acid sequence was then used for BLAST in the zebrafish amino acid sequence database, and the DrCIG30L4 sequences were the most “matched” to each other. A ClustalW alignment and phylogenetic analysis of the zebrafish CIG30 / ELOV family also showed that HsELOVL4 and DrCIG30L4 have the closest relationship.

  Without limitation, a link between ELOVL4 and branching morphogenesis has been found. As mentioned above, ELOVL4 is associated with autosomal dominant Stargardt-like macular dystrophy (STDG3). Although no direct link was formed between STGD3, ELOVL4 and angiogenesis, PMO results in zebrafish suggest that there may be a potential link between angiogenic disorders and STGD3-related phenotypes Showed sex.

  Thus, ELOVE4 was identified as an interesting target drug for the treatment of pathologies associated with branching morphogenesis such as angiogenesis, particularly those associated with vascular p53 signaling.

Claims (32)

(A) providing an assay system comprising a purified LCE polypeptide or nucleic acid, or a functionally active fragment or derivative thereof;
(B) contacting the assay system with the test agent under conditions such that the system provides control activity in the absence of the test agent;
(C) detecting the activity of the assay system affected by the test agent and identifying the test agent as a candidate p53 pathway modulator by the difference between the test agent affected activity and the control activity A method of identifying a p53 pathway modulator.   2. The method of claim 1, wherein the assay system comprises cultured cells that express the LCE polypeptide.   The method of claim 2, wherein the cultured cells further have defective p53 function.   2. The method of claim 1, wherein the assay system comprises a screening assay comprising an LCE polypeptide, and the candidate test agent is a small molecule modulator.   5. The method of claim 4, wherein the assay is a binding assay.   The method of claim 1, wherein the assay system is selected from the group consisting of an apoptosis assay system, a cell proliferation assay system, an angiogenesis assay system, and a hypoxia induction assay system.   2. The method of claim 1, wherein the assay system comprises a binding assay comprising an LCE polypeptide and the candidate test agent is an antibody.   The method of claim 1, wherein the assay system comprises an expression assay comprising LCE nucleic acid and the candidate test agent is a nucleic acid modulator.   9. The method of claim 8, wherein the nucleic acid modulator is an antisense oligomer.   9. The method of claim 8, wherein the nucleic acid modulator is PMO. (D) The candidate p53 pathway regulator identified in (c) is administered to a model system containing cells defective in p53 function, and a phenotypic change in the model system indicating that p53 function is restored is detected The method of claim 1, further comprising:   The method according to claim 11, wherein the model system is a mouse model having defective p53 function.   contacting cells defective in p53 function with a candidate modulator that specifically binds to an LCE polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, and 16. A method of modulating the p53 pathway of a cell, wherein the p53 function is restored.   14. The method of claim 13, wherein the candidate modulator is administered to a vertebrate that has been previously determined to have a disease or disorder resulting from a defect in p53 function.   14. The method of claim 13, wherein the candidate modulator is selected from the group consisting of an antibody and a small molecule. (D) providing a secondary assay system comprising a non-human animal or cultured cells expressing LCE;
(E) a secondary assay system is contacted with the test agent of (b) or an agent derived therefrom under conditions that provide a control activity by the secondary assay system in the absence of the test agent or an agent derived therefrom. Steps,
(F) detecting the activity of the secondary assay system affected by the agent, and the test agent or the derivative derived from the difference between the activity of the secondary assay system affected by the agent and the control activity The identified agent is a candidate p53 pathway modulator,
2. The method of claim 1, wherein the second assay detects a change in the p53 pathway affected by the agent.   The method of claim 16, wherein the secondary assay system comprises cultured cells.   The method of claim 16, wherein the secondary assay system comprises a non-human animal.   19. The method of claim 18, wherein the non-human animal misexpresses a gene for the p53 pathway.   A method of modulating the p53 pathway in a mammalian cell, comprising contacting the mammalian cell with an agent that specifically binds to an LCE polypeptide or nucleic acid.   21. The method of claim 20, wherein the agent is administered to a mammal previously determined to have a pathology associated with the p53 pathway.   21. The method of claim 20, wherein the agent is a small molecule modulator, a nucleic acid modulator, or an antibody. (A) obtaining a biological sample from a patient;
(B) contacting the sample with a probe for LCE expression;
(C) comparing the results from step (b) with a control; and (d) determining whether step (c) indicates a possible disease, diagnosing the patient's disease.   24. The method of claim 23, wherein the disease is cancer.   25. The method of claim 24, wherein the cancer is a cancer shown in Table 1 as having an expression level of> 25%. (A) providing an assay system comprising an ELOVL4 polypeptide or nucleic acid;
(B) contacting the assay system with the test agent under conditions where the system provides a control activity in the absence of the test agent; and (c) the activity of the assay system affected by the test agent. Detecting and identifying the test agent as a branched morphogenesis candidate modulator by the difference between the activity affected by the test agent and the control activity.   27. The method of claim 26, wherein the assay system comprises a non-human animal or cultured cell that expresses ELOVL4, and the assay system comprises an assay that detects modulation affected by the test agent in branched morphogenesis.   28. The method of claim 27, wherein the branching morphogenesis is angiogenesis.   28. The assay system comprising cultured cells, wherein the assay detects a phenomenon selected from the group consisting of cell proliferation, cell cycle, apoptosis, capillary formation, cell migration, cell sprouting and response to hypoxia. The method described.   28. The method of claim 27, wherein the assay system comprises a non-human animal and the assay system comprises a Matrigel assay or a xenograft assay. (D) providing a secondary assay system comprising a non-human animal or cultured cells that express ELOVL4;
(E) contacting the secondary assay system with the test agent of (b) or an agent derived therefrom under conditions such that in the absence of the test agent or test agent derived therefrom, the system provides control activity. And (f) detecting the activity of the secondary assay system affected by the agent, and is derived from the difference between the agent-affected activity and the control activity of the secondary assay system. Confirming the drug as a branching morphogenesis candidate modulator,
27. The method of claim 26, wherein the secondary assay system comprises a second assay that detects an agent-affected modulation in activity associated with branching morphogenesis.   A method of modulating branched morphogenesis of a mammalian cell, comprising contacting the cell with an agent that specifically binds to an ELOVL4 polypeptide or nucleic acid.