WO2001010883A1 - Cell death gene and uses thereof - Google Patents

Abstract

The invention features a novel gene, PERP, which is downstream of p53 in the p53-dependent apoptosis pathway, and which, when over-expressed, induces apoptosis. PERP may also be involved in p53-independent apoptosis. Stimulation of PERP expression or mimicry of its activity provides a mechanism for re-sensitizing tumor cells to radiation- and chemotherapy-induced apoptosis. Conversely, inhibition of PERP may be used to inhibit pathological cell death, e.g., in neural, cardiac, and retinal degenerative disease.

Description

CELL DEATH GENE AND USES THEREOF

Field of the Invention The field of the invention is regulation of cell death.

Background of the Invention p53 is a DNA binding protein and transcriptional activator that functions as a tumor suppressor protein. Up-regulation of p53 expression induces either cell cycle arrest or apoptosis, and cells having p53-inactivating mutations are resistant to these mechanisms of growth control. Strikingly, at least 50% of sporadic human cancers contain mutations that inactivate p53. Moreover, germline transmission of such mutations is responsible for the inheritance of cancer susceptibility, for example, Li-Fraumeni syndrome, in which affected individuals develop a variety of cancers at an early age. p53 acts as a tumor suppressor by inducing either growth arrest or apoptosis in cells with damaged DNA. Each of these pathways appears to involve p53-dependent transcriptional activation of a distinct gene expression program. For example, exposure of normal cells to DNA-damaging agents stimulates p53-dependent transcription of cell cycle arrest genes, such as p21/wafl/cipl and mdm2. p53-induced growth arrest provides an opportunity for DNA repair before replication and cell division, thereby lessening the chance that mutations will be transmitted to daughter cells.

By contrast, growth-deregulated cells, such as cells that constitutively express oncogenes (experimental models include adenovirus El A, SV40 T-antigen, and c-Myc) do not undergo p53-dependent Gl arrest. Instead, such cells normally undergo p53-dependent apoptosis. However, oncogene-expressing cells that also contain inactivating p53 mutations (e.g., some cancer cells) are resistant to both p53-mediated Gl arrest and apoptosis, and, as a result, proliferate uncontrollably. Accordingly, the presence of p53 mutations in cancer cells is a predictor of responsiveness to anti-tumor therapy: in contrast to cancer cells that express functional p53, cancer cells that do not express functional p53 are relatively resistant to chemical- and radiation- induced apoptosis.

The p53 -dependent apoptotic pathway is poorly understood, and few p53 transcriptional target genes have been identified. Given that the efficacy of many non-surgical cancer treatments is dependent upon p53-mediated apoptosis, it is likely that identification of apoptosis genes downstream of p53 will facilitate the development of novel strategies for re-sensitizing p53- mutated tumor cells to chemotherapy and radiation therapy. Re-sensitization therapy, in conjunction with conventional cancer therapy, could greatly enhance the efficacy of treatment for many cancer patients. p53 also plays a role in apoptosis of non-tumor cells. For example, neurons, cardiomyocytes, epidermal cells, and retinal photoreceptor cells, undergo p53-dependent apoptosis, particularly under pathological conditions. Therefore, novel approaches for inhibiting p53-dependent apoptosis are likely to provide new therapies for a broad variety of degenerative diseases and injuries that involve pathological cell death.

Summary of the Invention We have used subtractive hybridization to isolate a novel gene that is transcriptionally up-regulated during p53-dependent apoptosis. Overexpression of this gene, which we have denoted PERP (£53 apoptosis effector related to PMP-22), also known as PAAT-1 (£53 apoptosis-associated target I), is sufficient to induce apoptosis in cultured fibroblasts. Moreover, PERP is expressed in p53-negative cells undergoing TNF-α-induced apoptosis, suggesting that PERP may also be involved in p53-independent apoptosis.

In a first aspect, the invention features a substantially pure PERP polypeptide. In preferred embodiments of the first aspect of the invention, the PERP polypeptide is a mammalian polypeptide, such as a mouse or human polypeptide, and the polypeptide includes an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 11, such as the amino acid sequence shown in Fig. 11 (SEQ ID NO: 2).

In a second aspect, the invention features a substantially pure nucleic acid that includes a nucleotide sequence that encodes a PERP polypeptide. In preferred embodiments of the second aspect of the invention, the nucleic acid includes a nucleotide sequence that encodes a mammalian PERP polypeptide, such as a mouse or human PERP polypeptide, and the nucleic acid includes a nucleotide sequence that encodes a PERP polypeptide containing an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 11, such as the amino acid sequence shown in Fig. 11. The substantially pure nucleic acid may include the PERP coding sequence shown in Fig. 10 (SEQ ID NO: 1). In addition, the nucleic acid may be within an expression vector, wherein the nucleic acid is operably linked to a promoter.

In a third aspect, the invention features a non-human transgenic animal, wherein the animal contains a substantially pure nucleic acid including a nucleotide sequence that encodes a PERP polypeptide. In a preferred embodiment of the third aspect of the invention, the non-human transgenic animal may be a mouse. In a fourth aspect, the invention features a non-human animal, wherein one or both endogenous alleles encoding PERP are mutated, disrupted, or deleted in the non-human animal. The non-human animal may be a mouse, pig, goat, sheep, or cow. In addition, the mutation, disruption, or deletion of the endogenous alleles encoding PERP may be conditional, such that PERP expression is spatially or temporally controlled.

In a fifth aspect, the invention features a substantially pure nucleic acid that includes at least 14 consecutive nucleotides that display at least 85%, 90%, 92%, 95%, or 98% sequence identity to a nucleotide sequence that is complementary to a PERP nucleic acid. In preferred embodiments of the fifth aspect of the invention, the nucleic acid includes at least 16, 18, 22, 25, 50, 75, or 100 consecutive nucleotides that display at least 85%, 90%, 92%, 95%, or 98% sequence identity to a nucleotide sequence that is complementary to a PERP nucleic acid, and the nucleic acid hybridizes under high stringency conditions to a PERP nucleic acid.

In a sixth aspect, the invention features a substantially pure nucleic acid including at least 14 nucleotides, wherein the nucleic acid hybridizes under high stringency conditions to a PERP nucleic acid. In a preferred embodiment of the sixth aspect of the invention, the substantially pure nucleic acid includes at least 16, 18, 22, 25, 50, 75, or 100 nucleotides.

In another embodiment of the fifth and sixth aspects of the invention, the substantially pure nucleic acid is an antisense nucleic acid.

In a seventh aspect, the invention features a method of detecting the presence of a PERP nucleic acid. The method includes (a) contacting a sample, under high stringency conditions, with a nucleic acid probe, wherein the probe hybridizes under high stringency conditions to a PERP nucleic acid; and (b) assaying for the presence of hybridized probe, wherein the presence of hybridized probe indicates the presence of a PERP nucleic acid.

In a preferred embodiment of the seventh aspect of the invention, the method is for detecting PERP biological activity; the PERP nucleic acid is genomic DNA, cDNA, or mRNA; and the method further includes use of the polymerase chain reaction (PCR), wherein the presence of a PCR product indicates the presence of the PERP nucleic acid.

In an eighth aspect, the invention features a method of inhibiting expression of PERP in a cell. The method includes introducing into the cell a PERP antisense nucleic acid.

In a ninth aspect, the invention features a method for stimulating apoptosis in a population of cells. The method includes introducing into the cells substantially pure PERP polypeptide, wherein the PERP polypeptide stimulates apoptosis in the cells, compared to cells not containing the PERP polypeptide.

In a preferred embodiment of the ninth aspect of the invention, the PERP polypeptide is encoded by a substantially pure PERP nucleic acid, wherein the nucleic acid is introduced into the cells.

In various preferred embodiments of the eighth and ninth aspects of the invention, the cells are tumor cells; the cells are exposed to an apoptotic stimulus before or after the PERP polypeptide is introduced into the cells; and the apoptotic stimulus is gamma irradiation or a chemotherapeutic agent.

In a tenth aspect, the invention features a method for inhibiting apoptosis in a population of cells having an increased risk for undergoing apoptosis. The method includes introducing into the cells a PERP antisense nucleic acid, wherein the PERP antisense nucleic acid decreases the level of PERP in the cells, and wherein the decrease inhibits apoptosis in the cells.

In preferred embodiments of the tenth aspect of the invention, the increased risk for undergoing apoptosis is caused by: exposure to gamma irradiation, exposure to a chemotherapeutic agent, exposure to a toxin, exposure to hypoxia, an injury, a degenerative disease, or an attack by cells of the immune system.

In an eleventh aspect, the invention features a method of identifying a compound that modulates apoptosis. The method includes the steps of: (a) exposing a sample to a test compound, wherein the sample includes a PERP nucleic acid, a PERP reporter gene, or a PERP polypeptide; and (b) assaying for a change in the level of PERP biological activity in the sample, relative to a sample not exposed to the test compound, wherein an increase in the level of PERP biological activity in the sample indicates a compound that stimulates apoptosis, and a decrease in the level of PERP biological activity in the sample indicates a compound that inhibits apoptosis.

In preferred embodiments of the eleventh aspect of the invention, the PERP nucleic acid is genomic DNA, cDNA, mRNA, cRNA, or a substantially pure genomic DNA fragment, and the PERP nucleic acid, PERP reporter gene, or PERP polypeptide is within a cell, wherein the cell is exposed to a test compound.

In a twelfth aspect, the invention features an antibody that specifically binds PERP. In a preferred embodiment of the twelfth aspect of the invention, the antibody is a monoclonal antibody or a polyclonal antibody. In a thirteenth aspect, the invention features a method of detecting the presence of a PERP polypeptide. The method includes: (a) contacting a sample with an antibody that specifically binds the PERP polypeptide; and (b) assaying for the binding of the antibody to the PERP polypeptide, wherein the binding of the antibody to the PERP polypeptide indicates the presence of a PERP polypeptide. In a preferred embodiment of the thirteenth aspect of the invention, the method is for detecting PERP biological activity.

In a fourteenth aspect, the invention features a substantially pure nucleic acid that comprises at least 14 consecutive nucleotides that display at least 85%, 90%, 92%, 95%, or 98% sequence identity to a nucleotide sequence within a PERP nucleic acid, wherein the substantially pure nucleic acid is not an expressed sequence tag set forth in Table 1 or Table 2 (SEQ ID NOS: 16- 259). The substantially pure nucleic acid may be used, for example, as a probe to detect PERP genomic DNA or cDNA. The substantially pure nucleic acid may also be used as an antisense nucleic acid to inhibit transcription of a PERP gene.

By "apoptosis" is meant a cell death pathway wherein a dying cell displays a set of well-characterized biochemical hallmarks that include cytolemmal membrane blebbing, cell soma shrinkage, chromatin condensation, nuclear disintegration, and DNA laddering. There are many well-known assays for determining the apoptotic state of a cell, including, and not limited to: reduction of MTT tetrazolium dye, TUNEL staining, Annexin V staining, propidium iodide staining, DNA laddering, PARP cleavage, caspase activation, and assessment of cellular and nuclear morphology. Any of these or other known assays may be used in the methods of the invention to determine whether cells are undergoing apoptosis. By "increased risk for undergoing apoptosis" is meant a population of cells that is exposed to an apoptotic stimulus, e.g., gamma irradiation, a chemotherapeutic agent, a toxin, an injury, an attack by cells of the immune system, hypoxia, or a degenerative disease. Apoptosis is stimulated in such a cell population by at least 5%, preferably by at least 10%, more preferably by at least 25%, still more preferably by at least 50%, and most preferably by at least 75%.

By "stimulating apoptosis" is meant increasing the number of apoptotic cells in a population of cells by at least 5%, preferably by at least 10%, more preferably by at least 25%, still more preferably by at least 50%, and most preferably by at least 75%.

By "inhibiting apoptosis" is meant decreasing the number of apoptotic cells in a population of cells by at least 1% to 5%, preferably by at least 10%, more preferably by at least 25%, still more preferably by at least 50%, and most preferably by at least 75%.

By "PERP polypeptide" is meant a polypeptide that induces apoptosis and contains an amino acid sequence that bears 80% sequence identity, preferably at least 85% sequence identity, more preferably at least 90% sequence identity, still more preferably at least 95% sequence identity, and most preferably 100% sequence identity with the PERP amino acid sequence shown in Fig. 11.

By "PERP nucleic acid" is meant an RNA (e.g., an mRNA) or DNA (e.g., a genomic DNA, a genomic DNA fragment, or a cDNA) molecule that encodes PERP.

By "PERP coding sequence" is meant a nucleotide sequence that encodes a PERP polypeptide. By "PERP biological activity" is meant the ability of the PERP polypeptide to induce apoptosis. The level of PERP biological activity, e.g., death-inducing activity, may be directly measured using any of the many known assays for measuring apoptosis. In addition, the relative level of PERP biological activity may also be assessed by measuring the level of PERP mRNA (e.g., by reverse transcription-poly merase chain reaction (RT-PCR) amplification or Northern hybridization), the level of PERP protein (e.g., by ELISA or Western hybridization), the activity of a reporter gene under the transcriptional regulation of a PERP transcriptional regulatory region (by reporter gene assay, as described below), or the specific interaction of PERP with another molecule (e.g., by the two-hybrid assay); e.g., but not limited to, a polypeptide that is activated by PERP or that inhibits PERP activity. For example, a compound that increases the level of PERP mRNA, protein, or reporter gene activity within a cell, a cell extract, or other experimental sample is a compound that stimulates the biological activity of PERP.

By "endogenous" is meant a nucleic acid, polypeptide, or protein that is naturally present within a cell, i.e., not artificially introduced by genetic engineering.

By "PERP antisense nucleic acid" is meant a nucleic acid complementary to a PERP coding sequence. Preferably, the antisense nucleic acid decreases expression (i.e., transcription and/or translation) of PERP by at least 5%, more preferably by at least 10%, still more preferably by at least 20% to 30%, and most preferably by at least 50% to 70%. Preferably, a PERP antisense nucleic acid comprises from about 8 to 30 nucleotides. A PERP antisense nucleic acid may also contain at least 40, 60, 85, 120, or more consecutive nucleotides that are complementary to a PERP mRNA or DNA, and may be as long as a full-length PERP gene or mRNA. The antisense nucleic acid may contain a modified backbone, for example, phosphorotioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.

A PERP antisense nucleic acid may also be in a vector where the vector is capable of directing expression of the antisense nucleic acid. This vector may be inserted into a cell using methods known to those skilled in the art. For example, the full length PERP nucleic acid sequence, or portions thereof, can be cloned into a retroviral vector and driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest. Other viral vectors which can be used include adenovirus, adeno-associated virus, vaccinia virus, bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus.

By "expose" is meant to allow contact between an animal, cell, lysate or extract derived from a cell, or molecule derived from a cell, and a test compound or apoptotic stimulus.

By "treat" is meant to submit or subject an animal, cell, lysate or extract derived from a cell, or molecule derived from a cell to a test compound or apoptotic stimulus.

By "test compound" is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate cell death or PERP biological activity, by employing one of the assay methods described herein. Test compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof. By "assaying" is meant analyzing the effect of a treatment, be it chemical or physical, administered to whole animals, cells, or molecules derived therefrom. The material being analyzed may be an animal, a cell, a lysate or extract derived from a cell, or a molecule derived from a cell. The analysis may be, for example, for the purpose of detecting altered gene expression, altered RNA stability, altered protein stability, altered protein levels, or altered protein biological activity. The means for analyzing may include, for example, antibody labeling, immunoprecipitation, phosphorylation assays, and methods known to those skilled in the art for detecting nucleic acids.

By "sample" is meant an animal, a cell, a lysate or extract derived from a cell, or a molecule derived from a cell or cellular material, which is assayed as described above.

By "modulating" is meant changing, either by decrease or increase.

By "a decrease" is meant a lowering in the level of: a) protein (e.g., as measured by ELISA); b) reporter gene activity (e.g., as measured by reporter gene assay, for example, lacZ/β-galactosidase, green fluorescent protein, or luciferase activity); c) mRNA (e.g., as measured by RT-PCR relative to an internal control, for example, a "housekeeping" gene product such as β-actin or glyceraldehyde 3 -phosphate dehydrogenase (GAPDH)); or d) the number of apoptotic cells in a test sample. In all cases, the lowering is preferably by at least 30%, more preferably by at least 40% to 60%, and even more preferably by at least 70%.

By "an increase" is meant a rise in the level of: a) protein (e.g., as measured by ELISA); b) reporter gene activity (e.g., as measured by reporter gene assay, for example, lacZ/β-galactosidase, green fluorescent protein, or luciferase activity); c) mRNA (e.g., as measured by RT-PCR relative to an internal control, for example, a "housekeeping" gene product such as β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) or d) the number of apoptotic cells in a test sample. Preferably, the increase is by at least 1.5-fold to 2-fold, more preferably by at least 3-fold, and most preferably by at least 5- fold.

By "alteration in the level of gene expression" is meant a change in transcription, translation, or mRNA or protein stability such that the overall amount of a product of the gene, i.e., mRNA or polypeptide, is increased or decreased.

By "reporter gene" is meant any gene that encodes a product whose expression is detectable and/or quantitatable by immunological, chemical, biochemical or biological assays. A reporter gene product may, for example, have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ/β-galactosidase, luciferase, chloramphenicol acetyltransferase), toxicity (e.g., ricin A), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably labelled antibody). It is understood that any engineered variants of reporter genes, which are readily available to one skilled in the art, are also included, without restriction, in the foregoing definition.

By a "transgene" is meant a nucleic acid sequence that is inserted by artifice into a cell and becomes a part of the genome of that cell and its progeny. Such a transgene may be (but is not necessarily) partly or entirely heterologous (e.g., derived from a different species) to the cell.

By "transgenic animal" is meant an animal comprising a transgene as described above. By "knockout mutation" is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% relative to the unmutated gene. The mutation may, without limitation, be an insertion, deletion, frameshift, or missense mutation. A "knockout animal," e.g. a knockout mouse, is an animal containing a knockout mutation. The knockout animal may be heterozygous or homozygous for the knockout mutation. The knockout animal may also be a conditional knockout, in which the mutation is temporally or spatially controlled.

By "protein" or "polypeptide" or "polypeptide fragment" is meant any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide.

By "cRNA" is meant an RNA molecule produced by an in vitro transcription reaction, e.g., using a plasmid template that encodes the cRNA. cRNAs may be produced by well-known methods; for example, Promega Corp. (Madison, WI) produces in vitro transcription kits, e.g., Riboprobe® and RiboMAX™.

By "operably linked" is meant that a gene and one or more transcriptional regulatory sequences, e.g., a promoter or enhancer, are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences. By "promoter" is meant a minimal sequence sufficient to direct transcription of a gene. Also included in this definition are those transcription control elements (e.g., enhancers) that are sufficient to render promoter- dependent gene expression controllable in a cell type-specific, tissue-specific, or temporal-specific manner, or inducible by external signals or agents; such elements, which are well-known to skilled artisans, may be found in a 5' or 3' region of a gene or within an intron.

By "expression vector" is meant a DNA construct that contains a promoter operably linked to a downstream gene or coding region (e.g., a cDNA or genomic DNA fragment, which encodes a protein, or an antisense RNA coding region). Transfection of the expression vector into a recipient cell allows the cell to express RNA encoded by the expression vector. An expression vector may be a genetically engineered plasmid, virus, or artificial chromosome derived from, for example, a bacteriophage, adenovirus, retrovirus, poxvirus, or herpesvirus.

By "identity" is meant that a polypeptide or nucleic acid sequence possesses the same amino acid or nucleotide residue at a given position, compared to a reference polypeptide or nucleic acid sequence to which the first sequence is aligned. Sequence identity is typically measured using sequence analysis software with the default parameters specified therein, such as the introduction of gaps to achieve an optimal alignment. Examples of programs for sequence analysis include the Wisconsin Package™, Version 10.0 (Genetics Computer Group, Madison, WI; http://www.gcg.com) or the Basic Local Alignment Search Tool (BLAST) available form the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST). By "substantially identical" is meant a polypeptide or nucleic acid exhibiting, over its entire length, at least 40%, preferably at least 50%-85%, more preferably at least 90%, and most preferably at least 95%-99% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences is at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids. For nucleic acids, the length of comparison sequences is at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 110 nucleotides.

By "substantially pure polypeptide" is meant a polypeptide (or a fragment thereof) that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is a PERP polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure PERP polypeptide may be obtained, for example, by extraction from a natural source (e.g., a mammalian cell), by expression of a recombinant nucleic acid encoding a PERP polypeptide (e.g., in a cell or in a cell-free translation system), or by chemically synthesizing the polypeptide. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides not only include those derived from eukaryotic organisms but also those synthesized in E. coli or other prokaryotes.

By "substantially pure DNA" is meant DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

By "high stringency conditions" is meant conditions that allow hybridization comparable with those resulting from the use of a DNA probe of at least 40 nucleotides in length, in a buffer containing 0.5 M NaHP0₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65° C, or a buffer containing 48% formamide, 4.8X SSC, 0.2 M Tris-Cl, pH 7.6, IX Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42° C. Other conditions for high stringency hybridization, such as for PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well- known by those skilled in the art of molecular biology. See, e.g., F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998, hereby incorporated by reference.

By "specifically binds" is meant a molecule, e.g., an antibody, that recognizes and binds a PERP polypeptide, particularly a mammalian PERP polypeptide (such as a human or murine PERP polypeptide), but that does not substantially recognize and bind other non-PERP molecules in a sample, e.g., a biological sample, that naturally includes protein.

By "transformation" or "transfection" is meant any method for introducing foreign molecules into a cell (e.g., a bacterial, yeast, fungal, algal, plant, insect, or animal cell, particularly a mammalian cell). Lipofection, DEAE-dextran-mediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, viral delivery, electroporation, and biolistic transformation are just a few of the methods known to those skilled in the art which may be used.

By "transformed cell" or "transfected cell" is meant a cell (or a descendent of a cell) into which a DNA molecule encoding a PERP polypeptide of the invention has been introduced, by means of recombinant DNA techniques. Such cells may be either stably or transiently transfected.

"Translation" as used herein and as used by those of skill in the art, refers to the process of generating a polypeptide that has an amino acid sequence dictated by the codon sequence of an mRNA that encodes the polypeptide.

"Transcription" as used herein and as used by those of skill in the art, refers to the process of using a DNA sequence as a template to generate a messenger RNA (mRNA) molecule of given nucleotide sequence.

Brief Description of the Drawings

Fig. 1 is a diagram showing that p53 induces Gl arrest and apoptosis by distinct mechanisms.

Fig. 2 is a diagram showing the subtractive hybridization strategy used to identify genes involved in p53-dependent apoptosis. Fig. 3 is a diagram of a Northern blot showing PERP expression in adriamycin-treated p53+/+ (wild-type), EIA p53-r-/+, and EIA p53-/- mouse embryonic fibroblasts (MEFs).

Fig. 4 is a diagram of a Northern blot showing a time course of PERP expression in p53-r-/+ (wild-type), EIA p53+/+, and EIA p53-/- MEFs undergoing adriamycin-induced Gl arrest or apoptosis.

Fig. 5 is a diagram of a Northern blot showing a time course of PERP expression in p53+/+ (wild-type) and p53-/- MEFs undergoing ultraviolet light-induced apoptosis.

Fig. 6 is a diagram of a Northern blot showing PERP expression in

EIA ras p53-/- MEFs undergoing TNF-α-induced, p53-independent apoptosis.

Fig. 7 is a diagram of the strategy for studying the effect of PERP overexpression in NIH 3T3 cells.

Fig. 8 is a diagram of an immunocytofluorescence assay showing that HA-tagged PERP localizes to cytoplasmic structures in NIH 3T3 cells.

Fig. 9 is a diagram of an immunocytofluorescence assay showing that expression of HA-tagged PERP induces apoptosis in NIH 3T3 cells.

Fig. 10 is a diagram showing the nucleotide sequence of a PERP cDNA (SEQ ID NO: 1).

Fig. 11 is a diagram showing the predicted amino acid sequence of

PERP (SEQ ID NO: 2) and its alignment with PMP-22 and PMP-22-2 from human, mouse, and rat, and a consensus sequence (SEQ ID NOS: 3-8).

Fig. 12 is a diagram showing the hydropathy profile of PERP. Detailed Description of the Invention

p53 is a DNA binding protein and transcriptional activator that inhibits tumor formation by two discrete mechanisms: induction of growth arrest or induction of apoptosis. Functional p53 is required for susceptibility of cancer cells (which are growth-deregulated, and therefore, do not readily undergo Gl arrest) to apoptotic stimuli such as gamma irradiation and chemotherapeutic agents. p53 also appears to be involved in disease- and injury-induced apoptosis of non-tumor cells. Efforts to identify genes that are specific downstream targets of p53 in the p53-dependent apoptotic pathway have been largely unsuccessful, and the mechanism underlying p53-dependent apoptosis remains poorly understood.

We designed a subtractive hybridization strategy for isolating genes that are downstream targets of p53 and that are specifically expressed in apoptosis versus Gl arrest. Our strategy was based on the previous observation that MEFs treated with DNA-damaging agents undergo p53-dependent Gl arrest, whereas growth-deregulated MEFs expressing the adeno virus EIA oncoprotein and treated with DNA-damaging agents undergo p53-dependent apoptosis (Fig. 1). To isolate cDNAs encoding proteins involved in apoptosis, we performed subtractive hybridization cloning using "driver" cDNA and "tester" cDNA from MEFs treated with the DNA-damaging agent adriamycin (Fig. 2). Tester cDNA was prepared from p53+/+ MEFs expressing the adenoviral protein EIA (EIA p53-r-/+); these cells undergo apoptosis when treated with adriamycin. Driver cDNA was prepared from two types of MEFs: p53+/+ MEFs (not expressing EIA), which undergo Gl arrest in response to adriamycin treatment, and p53-/- MEFs expressing EIA (EIA p53-/-), which, in contrast, undergo neither Gl arrest nor apoptosis in response to adriamycin treatment. The driver cDNA from Gl -arrested p53+/+ MEFs was used to eliminate from the tester population any transcripts that are up-regulated by p53 during both Gl arrest and apoptosis. The driver cDNA from EIA p53-/- MEFs was used to eliminate any mRNAs that are induced by EIA protein in a p53- independent manner. Hybridization with driver cDNA also eliminates "housekeeping" transcripts (e.g., those encoding β-actin) common to both mRNA populations.

To enrich the tester population for cDNAs encoding proteins that are specifically involved in p53-dependent apoptosis, an excess of single-stranded driver cDNA (from Gl -arrested p53+/+ MEFs and non-arrested, non-apoptotic p53-/- MEFs) was hybridized with tester cDNA (from apoptotic EIA p53+/+ MEFs). The single-stranded cDNAs that were unique to the tester population, i.e., cDNAs from genes that are transcriptionally up-regulated during p53- dependent apoptosis, were amplified by the polymerase chain reaction (PCR) using primers corresponding to linkers attached to the cDNAs.

Our screen yielded 56 partial cDNAs. DNA sequence analysis showed that 8 of the clones were represented 2-4 times and that approximately 21 of the clones were represented once. Most of the clones corresponded to known genes, some were novel, and some corresponded to expressed sequence tags (ESTs) found in dbEST (http://www.ncbi.nlm.nih.gov/dbEST/index.html). Using partial cDNAs as probes for Northern or RNase protection analysis to confirm that the clones were specifically expressed in apoptotic cells, we observed that two of the clones, each of which was isolated twice, showed a dramatic pattern of transcriptional up-regulation that was specific for apoptotic cells. These clones represented two different regions of a novel gene that we have named "PERP." The PERP nucleotide sequence corresponds to a number of mouse and human ESTs (Tables 1 and 2; SEQ ID NOS: 16-259) derived from a variety of tissues, suggesting that PERP may be broadly expressed. Herein we show that PERP expression is up-regulated in cells undergoing p53- dependent apoptosis, and that over-expression of PERP is sufficient to induce apoptosis in NIH 3T3 cells. Moreover, we show that PERP is expressed at low levels in cells lacking p53 and undergoing p53 -independent apoptosis. Our data indicate that PERP may be used as a novel target for regulating apoptotic cell death.

Table 1: Genbank Accession Numbers for Mouse ESTs that Correspond to the PERP Nucleotide Sequence

AI553233. 1 AA238440 AI552218. 1 AI006601 AI314471

W64763 AI530500 AA080255 AA738986 AA986858

AA607915 AA638949 AI182192 AA469619 AI225641

W41075 AA866874 AA790615 AA030204 AA832815

W66664 AA031201 AA981808 AI227095 WO8900

W29886 AA028729 AA164119 AA563408 AI197174

AA162289 AA463132 AA498950 AA832772 AA169059

AA921197 AA500586 AA690252 W12639 AI049028

AA763130 AA164113 AA760474 AA790732 AV011540. .1

AA856086 AA930015 AV009123. ,1 AA529813 AI481983

AV084000. .1 AV023605. 1 AV081994. 1 AV014158. 1 AA098268

W30490 AA874469 AA038204 AI551433. 1 AI605141. .1

AV016545. .1 AV171063. .1 AA617030 AV079416. .1 AV023413. .1

AV079041. .1 W12529 AA213044 AV026760. .1 AV010395. .1

AV091872. .1 AV078192. .1 AI643260. .1 AV019165. ,1 AV019174. .1

AA272800 AV021246. ,1 AV167513. ,1 AA124625 AV084318. .1

AA124861 AV171331. ,1 AV012780. ,1 AV088340. ,1 AV010480. .1

AA038599 AA589534 AA492828 AA790733 AA209750

AV133488. .1 AV084623. .1 AV084375. .1 AA209741 AA185319

AI509295 AI552286. .1 AI645955. .1 AU067046. .1 W29526

AI553233. .1

Table 2: Genbank Accession Numbers for Human ESTs that Correspond to the PERP Nucleotide Sequence

H99748 R32216 R32172 AI278514 AI168626

AI022434 AA580370 AA160381 H97740 AI283412 AA158761 AA588751 R39395 R38317 AI475132

AA079807 AA079633 AI383070 R82584 R82585

AI346389 AA506029 AI146463 AI092688 93362

AI471235 R70905 R70906 93394 AA586975

AA159710 AA159711 H04495 H01942 AI167393 AI587086 N62092 AI291597 R63767 R63720

R70993 R70940 AA844033 AA482352 AA482254

H21696 H21906 R27158 R26913 R77169

AA618219 AI190841 AI075057 AA468385 AA468424

AI400768 AI561317 AI000172 AI422742 AI436339 AA843925 AA258741 AA258377 AI419413 AA583881

AI378931 AA932817 AI572472 R23770 R23723

AA480373 AI400366 AA471074 AA469417 AA631038

AI493331 AA253194 AA253195 AI492053 AI288461

AI004583 AI369025 AI202629 AI077673 AA614431 AA639961 N95055 AA906505 AI694126 AI682734

AI525592 AA296799 AA016243 AI683943 AA352092

AA626034 AI474703 AA385499 AA328654 AA654360

AI446687 AA186897 AI472890 AA298549 AA775509

AA935864 AI291596 AA016203 AA086218 AA328285 AA159815 AA610151 AA298285 T48546 AA297628

C17106 AA382912 AA297487 38893 AI740811

AA297236 T24990 AI572289 D79077 AI452382

C17487 AA298874 AI679511 AA688138 AA188520

AA159816 C00038 AI023936 AA568164 AA100383 AA076610 T10423 AA372564 AI436481 AF077755

AA297272 AA076609 N87013

General Methods

Preparation ofp53+/+ (Gl -arrested) andp53-X (non-arrested) MEFs

Wild-type (p53+/+) and homozygous p53 knockout (p53-/-) MEFs were isolated as described by Livingstone et al. Cell 70:923-935 (1992). To prepare Gl -arrested p53+/+ and p53-/- MEFs, 3.5-4.0 X 10⁶ cells per dish were seeded into five pi 00 (10-cm) tissue culture dishes for each of the two MEF genotypes. To synchronize the cells, they were first grown to confluence by culturing them in Dulbecco's Eagle Medium (DME) plus 10% fetal calf serum (FCS) for three days, after which they were cultured them in DME plus 0.1% serum for an additional four days. To stimulate re-entry into the cell cycle, the cells were re-seeded (in DME plus 10% FCS) at 1.0-1.6 X 10⁶ cells per dish into seventeen plOO dishes for each MEF genotype. After 6 hours, 0.2 μg/ml adriamycin (doxorubicin; Sigma, St. Louis, MO) was added to fifteen dishes of each genotype to induce Gl arrest. After 12 hours of adriamycin treatment, cells from thirteen dishes of each genotype were detached by trypsinization, centrifuged, and frozen as a pellet for subsequent RNA preparation. The remaining two dishes of treated cells and two dishes of untreated cells for each genotype were collected after 18 hours of adriamycin treatment and used for FACS analysis to verify that the p53+/+ MEFs were in Gl arrest, as described previously (Brugarolas et al. Nature 377:552-557, 1995). As expected, the p53-/- MEFs did not undergo Gl arrest, indicating that the arrest was p53- dependent.

Preparation of EIA p53+/+ (apoptotic) and EIA p53-X MEFs (non-apoptotic)

p53+/+ MEFs and p53-/- MEFs, both expressing adenovirus EIA (Samuelson et al. Proc. Nad. Acad. Sci. USA 94: 12094-12099, 1997) were obtained from Scott Lowe (Cold Spring Harbor Laboratory, Cold Spring

Harbor, NY). To generate apoptotic EIA p53+/+ MEFs and EIA p53-/- MEFs for RNA preparation, ten dishes of cells for each of the two genotypes were plated at about 2.5 X 10° cells per 10-cm dish. In addition, for apoptosis assays, 5 X 10⁴ cells per well were plated into each of six wells of a 24- well tissue culture dish for each of the two genotypes. All cells were treated with 0.2 μg/ml adriamycin.

For RNA preparation, the cells were collected by trypsinization after 18.5 hrs of treatment, a time at which approximately 22% of EIA p53+/+ cells and 3% of EIA p53-/- cells had undergone apoptosis. The amount of apoptosis was measured in the appropriate wells of the 24-well dishes at 15, 16, 17, 19, 24, and 42 hours after adriamycin treatment, by collecting and pooling both floating and adherent cells and determining the percentage of dead cells by trypan blue staining (live cells exclude trypan blue, and therefore are not stained, whereas dead cells accumulate the dye, and therefore become stained). The percentages of apoptotic cells were independently confirmed by staining the cells with 4',6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO) and counting the percentages of cells displaying characteristic apoptotic nuclear morphology (Attardi et al. EMBO J. 15:3693-3701, 1996).

Preparation ofMEF mRNA

Total RNA was prepared from growth-arrested, apoptotic, and non- arrested, non-apoptotic MEFs (having the genotypes described above and obtained as described above) using Ultraspec II (Biotecx, East Houston, TX) according to the manufacturer's instructions. Poly A⁺ RNA was isolated from total RNA using oligo-dT columns (Stratagene, La Jolla, CA) according to the manufacturer's instructions.

Subtractive hybridization cloning

Cloning by subtractive hybridization was performed using a PCR- Select™ cDNA Subtraction Kit (Clontech, Palo Alto, CA), according to the protocol supplied by the manufacturer. Tester cDNA was prepared using about 2 μg of polyA⁺ RNA from adriamycin-treated EIA p53+/+ MEFs. Driver cDNA was made from a 1 : 1 mixture of poly A⁺ RNA from two cell types: adriamycin-treated p53+/+ MEFs and adriamycin-treated EIA p53-/- MEFs (about 2 μg of polyA⁺ RNA from each cell type). Linkers provided in the kit were attached to the tester cDNA. The tester and driver cDNA were denatured and the tester cDNA was hybridized with an excess of driver cDNA to remove cDNAs that were common to both cDNA pools. cDNAs that were unique to the tester population were then amplified by polymerase chain reaction (PCR) using primers (provided in the kit) that were complementary to the linkers. The PCR-amplified cDNAs were TA-cloned into pT7Blue (Novagen, Madison, WI) and transformed into Escherichia coli XL-1 Blue or HB 101. Colonies were then subjected to differential screening analysis to eliminate false positives, as described by the Clontech protocol. Positives clones were subjected to DNA sequence analysis by dideoxy chain termination and mRNA expression analysis. Northern blots contained RNA from three types of adriamycin-treated MEFs: p53+/+ MEFs (which were Gl -arrested as a result of adriamycin treatment), EIA p53+/-r- MEFs (which were apoptotic as a result of adriamycin treatment) and EIA p53-/- MEFs (which were resistant to adriamycin-induced arrest and apoptosis). One particular clone ("PERP"), which was highly expressed in apoptotic cells relative to Gl -arrested cells, was selected for further analysis. Northern blot analysis

Equivalent amounts of RNAs were electrophoretically separated on formaldehyde gels and transferred to nylon filters using standard methods (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York, NY, 1998, or Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Pre-hybridization and hybridization was done using ExpressHyb Hybridization Solution (Clontech, Palo Alto, CA) according to the manufacturer's instructions. ³²P-radiolabeled probes (restriction fragments from cDNAs encoding PERP, GAPDH, cyclin G, or p21) were generated by radiolabeling 50-100 ng of the appropriate cDNA fragment using a random priming kit (Prime It Kit, Stratagene, La Jolla, CA) according to the manufacturer's instructions. To strip blots for re-probing, they were incubated 2 X 15 minutes in boiling water.

Northern blot (time course) analysis of RNA from adriamycin-treated cells

p53+/+ MEFs, EIA p53+/+ MEFs, and EIA p53-/- MEFs were plated on four pi 00 dishes for each cell type, and were left untreated (0 hour timepoint) or were treated for 2, 8, or 16 hours with 0.2 μg/ml adriamycin. At the 0, 2, 8, and 16 hour timepoints, the cells were collected and used for RNA preparation and Northern blot analysis as described above. The filter was hybridized first with a radiolabeled PERP probe, after which the filter was stripped and sequentially re-hybridized with radiolabeled probes for the p53 target genes cyclin G and p21 and the housekeeping gene GAPDH. Northern blot (time course) analysis of RNA from ultraviolet light-treated cells

p53+/-r- MEFs and p53-/- MEFs were plated at approximately 1.5-1.8 X 10⁶ cells per 10-cm dish. After 24 hours, apoptosis was induced by removing the medium from the cells and directly exposing them (i.e., without the lids of the cell culture dishes) to 20 J/m² of ultraviolet (UV) light using a Stratagene UV crosslinker. One plate of cells for each genotype was collected for RNA preparation at each of several timepoints after treatment (5, 10, 20, 29 hrs). One plate each of untreated p53+/+ MEFs and p53-/- MEFs was also collected for preparation of control RNA. The RNA samples were prepared and subjected to Northern analysis as described above.

Northern blot analysis of RNA from TNF-a-treated cells

p53-negative MEF cells expressing EIA and T24 H-ras (EIA ras p53-/- MEFs; Lowe et al. Proc. Natl. Acad. Sci. USA 91:2026-2030, 1994) were obtained from Scott Lowe (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Several million cells were plated into each of six pi 00 dishes and were either left untreated or were treated with 10 or 30 ng/ml TNF-α (Boehringer Mannheim, Indianapolis, IN) for 13 or 24 hrs. RNA was prepared at indicated timepoints (13 or 24 hours) and subjected to Northern analysis as described above. Cells were visibly undergoing apoptosis at the 24 hour timepoint.

Cloning and sequence analysis of full-length PERP cDNA

The full length PERP cDNA was isolated by 5' and 3' Rapid Amplification of cDNA Ends (RACE) using the Marathon cDNA amplification kit (Clontech, Palo Alto, CA), according to the manufacturer's protocol. Sequence analysis was performed using the BLAST

(http://www.ncbi.nlm.nih.gov/), Pfam (http://pfam.wustl.edu/), and MOTIF (http://www.public.iastate.edu/~pedro/motif_query.html) search programs.

Expression of PERP in mammalian cells

The PERP open reading frame (ORF) was amplified by polymerase chain reaction (PCR) using standard techniques (see, e.g., Ausubel et al., supra) and primers containing Sal I sites. The top strand primer consisted of the sequence

5'-TATGTCGACCTGCGCTGCGGCCTGGCCTG-3' (SEQ ID NO: 9) and the bottom strand primer consisted of the sequence 5'- CGCGTCGACGGCTGGGGGATAGAAGTACC-3' (SEQ ID NO: 10). The thermal cycler conditions were as follows: 1 cycle of 2 min at 94 °C, 30 sec at

50 °C, 2 min at 72 °C; 23 cycles of 40 sec at 94 °C, 30 sec at 50 °C, 2 min at 72 °C; and 23 cycles of 40 sec at 94 °C, 2 min at 72 °C. The PCR product was then cloned into the Sal I site of a derivative of the mammalian expression vector KA, which is a derivative of pcDNA3 (Invitrogen, Carlsbad, CA) that contains a DNA fragment encoding the influenza virus hemagglutinin (HA) tag. The resulting vector is KA/PERP-HA, which encodes PERP having an HA tag at its carboxyl terminus. The PERP expression construct was co-transfected with a β-galactosidase expression plasmid (as a marker for transfection) into NIH 3T3 cells using Lipofectamine™ (Gibco-BRL, Gaithersburg, MD) according to the manufacturer's instructions. Twenty-four to forty-eight hours after transfection, the transfected cells were stained by immunofluorescence as previously described (Attardi et al., supra), using a monoclonal anti-HA antibody at a dilution of 1: 1000 (Boehringer Mannheim, Indianapolis, IN; Catalog # 1583816), a polyclonal anti-β-galactosidase antibody at a dilution of 1 :50 (5'-3', Boulder, CO; Catalog # 5307-063100), and DAPI ( 1 μg/ml). FITC- coupled anti-mouse antibodies and rhodamine-coupled anti-rabbit antibodies were used to visualize HA and β-galactosidase, respectively. Cell death was assessed by examining the morphology of cells that were positive for PERP expression (dead cells appeared small and round with disintegrating or absent nuclei, as opposed to live cells, which appeared large and spread-out with intact nuclei).

PERP is up-regulated in MEFs undergoing p53-dependent apoptosis

To determine whether PERP is specifically up-regulated in cells undergoing apoptosis, we performed Northern blot analysis of RNA isolated from MEFs that had been treated with 0.2 μg/ml of the DNA-damaging agent adriamycin, as described in the Methods section. p53+/-r- (Gl -arrested) MEFs and EIA p53-/- (non-arrested, non-apoptotic; negative control) MEFs expressed low levels of PERP. In contrast, EIA p53+/+ (apoptotic) MEFs expressed high levels of PERP, suggesting that PERP is up-regulated during p53-dependent apoptosis (Fig. 3; the amount of GAPDH, a housekeeping gene, roughly indicates the relative amount of RNA in each lane). To gain a more detailed understanding of the temporal expression pattern of PERP expression during Gl arrest and apoptosis, we isolated RNA from p53+/+, EIA ρ53+/+, and El A p53-/- MEFs at 0, 2, 8, and 16 hours after adriamycin treatment and used Northern hybridization to assess the relative expression of PERP, compared to two genes that are known transcriptional targets of p53: cyclin G and p21.

Adriamycin-treated EIA p53-/- MEFs, which expressed very little p21 and cyclin G (Fig. 4 A, right), also expressed very little PERP, consistent with the hypothesis that the PERP gene, like the p21 and cyclin G genes, is a transcriptional target of p53. Adriamycin-treated p53 +/+ MEFs displayed a slight up-regulation in PERP expression only after extensive (16 hours) exposure to adriamycin (Fig. 4, left), and was probably due to high intracellular p53 accumulation. EIA p53+/-r- MEFs, which contain relatively high levels of p53 under basal conditions, also expressed relatively high basal levels of p21, cyclin G, and PERP, even in the absence of adriamycin treatment. PERP expression of these genes was up-regulated in response to adriamycin exposure, and continued to increase as the adriamycin exposure period increased (Fig. 4, middle). Taken together, these results indicate that PERP expression is up- regulated in cells undergoing p53-dependent apoptosis. Exposure of wild- type (p53-t-/+) MEFs to ultraviolet (UV) light, a DNA-damaging agent, stimulates p53 -dependent apoptosis and (to an equivalent degree but with delayed kinetics) p53-independent apoptosis. To determine whether PERP expression is up-regulated in a p53-dependent manner in cells undergoing UV light-induced apoptosis, we prepared mRNA from p53+/+ and p53-/- MEFs at 5, 10, 20, and 29 hours after their exposure to 20 J/m² of UV light. In parallel, we measured the percentage of apoptotic cells at these timepoints. Fig. 5 shows the results of Northern hybridization analyses for expression of PERP, GAPDH, and mdm-2 (a gene that is transcriptionally activated by p53 after DNA damage) at each timepoint ("UT" denotes untreated cells, i.e., cells not exposed to UV light). The bottom of the figure shows the percentage of apoptotic cells at each timepoint. For p53+/+ MEFs exposed to UV light (Fig. 5, left), mdm-2 and PERP expression increased as the percentage of apoptotic cells increased. By contrast, for p53-/- MEFs exposed to UV light (Fig. 5, right), expression of PERP and mdm-2 was barely detectable. p53-/- MEFs are far more resistant to UV light-stimulated apoptosis: at 20 hours after UV light exposure, only 4% of the p53-/- MEFs are apoptotic, compared to 21% of the p53+/+ MEFs (Fig. 5, bottom). Therefore, the above results are consistent with the hypothesis that PERP is involved in the p53-dependent apoptotic pathway.

PERP is not inducibly expressed in cells undergoing p53 -independent apoptosis

Expression of activated ras in growth-deregulated cells is often associated with an increased sensitivity to apoptosis. Tumor necrosis factor-α (TNF-α) is known to induce p53-independent apoptosis in MEFs expressing activated ras (see, e.g., Fernandez et al., Oncogene 9:2009-2017, 1994). To determine whether PERP is inducibly expressed in cells undergoing p53- independent apoptosis, we performed Northern analysis (Fig. 6) using RNA harvested from p53-/- MEFs expressing EIA and activated T24 H-ras (EIA ras p53-/- MEFs; Lowe et al. Proc. Natl. Acad. Sci. USA 91:2026-2030, 1994); prior to harvesting, the cells were treated with 20 ng/ml of TNF-α for 0, 2, 8, or 20 hours. RNA isolated from p53+/+ MEFs treated with adriamycin (indicated by a "+" in Fig. 6, last lane) was included as a positive control for p53- dependent PERP induction. After 20 hours of TNF-α treatment, at which 27% of the cells were apoptotic, PERP expression did not increase significantly, confirming that expression of PERP is induced by p53-dependent apoptosis. PERP is associated with cytoplasmic structures and induces apoptotic cell death

To determine the subcellular localization of PERP, we transfected an expression vector encoding HA-tagged PERP into NIH 3T3 cells and, 18 hours later, stained the cells using an antibody that specifically recognizes the HA tag. Immunofluorescence analysis revealed a punctate staining pattern within the cytoplasm and at the cell membrane, suggesting that PERP localizes to vesicles at these subcellular locations.

As described in the previous sections, we observed that PERP expression was up-regulated in cells undergoing p53-dependent cell death. Moreover, we noted that PERP mRNA was expressed, albeit at lower levels, in cells undergoing p53-independent apoptosis. To determine whether PERP alone is sufficient to induce apoptotic cell death, we co-transfected the expression vector encoding HA-tagged PERP into NIH 3T3 cells, along with a β-galactosidase expression vector as an internal control. As a negative control for cell death, we co-transfected NIH 3T3 cells with an empty expression vector plus the β-galactosidase expression vector. Twenty-four to forty-eight hours later, we stained the PERP-transfected cells with an anti-HA antibody and the control-transfected cells with an anti-β-galactosidase antibody. In addition, both samples were also stained with DAPI in order to assess nuclear integrity. The left panel of Fig. 9 shows that control cells expressing β- galactosidase alone appear well-attached with large, intact nuclei, i.e., they have the appearance of normal, healthy cells. In contrast, cells co-expressing PERP plus β-galactosidase appear small and round with dense, pyknotic nuclei, i.e., they have the appearance of apoptotic cells. This result indicates that expression of PERP alone can induce apoptotic cell death. PERP cDNA and its encoded polypeptide

The nucleic acid sequence of the PERP cDNA is shown in Fig. 10 (SEQ ID NO: 1; the ATG, indicating the initiator methionine, is underlined). Translation of this sequence yields a 193 amino acid polypeptide that bears limited sequence similarity to the peripheral myelin protein-22 (PMP-

22)/growth arrest-specific-3 (Gas-3) family of proteins (Fig. 11 ; SEQ ID NO: 2), which are involved in regulating growth arrest and apoptosis. Mutations in PMP-22 have been found in patients with Charcot-Marie-Tooth disease and other neuropathies.

We generated a hydropathy plot of the PERP polypeptide (Fig. 12) using Tmpred (http://www.ch.embnet.org/software/TMPRED_form.html). Tmpred makes a prediction of membrane-spanning regions and their orientation, using an algorithm based on the statistical analysis of TMbase, a database of naturally occurring transmembrane proteins. The hydropathy plot shows that PERP may contain four predicted transmembrane domains: 1) from residues 13 to 32 (intracellular to extracellular); 2) from residues 76 to 94 (extracellular to intracellular); 3) from residues 110 to 128 (intracellular to extracellular); and 4) from residues 151 to 171 (extracellular to intracellular).

PERP knockout animals

Mouse embryonic stem cells containing a triple-loxed PERP allele have been generated. The allele can be converted by partial or total Cre- mediated recombination to a conditional allele or to a null allele, respectively. These target ES cells can be used to generate transgenic mice containing the conditional or null PERP allele. Transgenic mice containing the conditional PERP allele can be intercrossed with transgenic mice that express Cre in a cell specific manner to obtain mice with PERP knocked out in a desired tissue according to standard methods known in the art, for example, Akagi et al. (Nucleic Acids Research 25: 1766-1773, 1997, or Giovannini et al. (Genes Dev. 14: 1617-30, 2000).

Stimulation of PERP expression or activity for cancer treatment

PERP is a downstream transcriptional target in the p53-dependent cell death pathway. Many tumors contain mutated p53 genes and, therefore, do not express functional p53. Such tumors are highly resistant to non-surgical anti-tumor therapies such as gamma irradiation and chemotherapy. Restoration of the p53-dependent cell death pathway would re-sensitize such tumor cells to these cancer treatments. We have shown that PERP is downstream of p53 in the cell death pathway and that PERP over-expression induces apoptosis. Therefore, one strategy for stimulating apoptosis in p53-negative tumor cells involves increasing the biological activity of PERP.

PERP biological activity in p53-deficient cells may be increased by various approaches. For example, intracellular PERP levels may be increased by administering, to a patient, an expression vector (such as a plasmid or viral vector) that contains a PERP coding region under the transcriptional regulation of a p53-independent promoter (e.g., a β-actin promoter or a viral promoter). The expression vector is administered such that it enters a target cell and expression of the vector-encoded PERP increases the susceptibility of the cell to apoptosis. Alternatively, a pharmaceutical agent that stimulates expression or activity of PERP in a p53-independent manner, or that mimics the biological activity of PERP, may be administered to the patient. These treatments may be targeted specifically to tumor cells by known methods, for example, using liposome delivery. However, these treatments need not be targeted to tumor cells: for example, at the appropriate level, a systemic increase in intracellular PERP activity may preferentially stimulate apoptosis in tumor cells and spare normal cells from a similar increased susceptibility to apoptosis.

We have shown that PERP is involved in p53-dependent apoptosis, that PERP is expressed in cells undergoing p53-independent apoptosis, and that overexpression of PERP alone is sufficient to induce apoptosis. Accordingly, PERP anti-cancer therapies may be used alone or in various therapeutic combinations, e.g., in conjunction with cancer therapies that induce apoptosis in a p53-dependent or p53 -independent manner, or with cancer therapies that do not induce apoptosis (e.g., surgery or cytostatic compounds). PERP therapy is useful for treating cancers that include, but are not limited to, cancers of the colon, pancreas, stomach, liver, skin (e.g., melanoma), lung, breast, ovary, cervix, uterus, bladder, testes, brain, and blood and lymphatic system (e.g., leukemias and lymphomas).

Inhibition of PERP expression or activity for treatment of p53-dependent degenerative diseases

p53-dependent apoptosis has been shown to occur in non-tumor cells undergoing degeneration associated with disease or injury. Such cells include, but are not limited to, cardiomyocytes, neurons, skin cells, and retinal photoreceptor cells. Accordingly, inhibition of the p53-dependent apoptotic pathway is useful for slowing the rate of (or halting) disease progression, or for lessening or preventing disease- or injury-associated tissue damage. One potential benefit of inhibiting the p53 -dependent apoptotic pathway by inhibiting PERP, rather than by inhibiting p53 itself, is that PERP appears to be preferentially expressed in apoptotic versus growth-arrested cells. Therefore, administration of a therapeutic agent that inhibits PERP may preferentially inhibit cell death. In contrast, administration of a therapeutic agent that inhibits p53 would inhibit not only cell death, but cell cycle arrest of DNA-damaged cells, thereby resulting in the accumulation of cells containing mutations, some of which might be tumorigenic. Inhibition of PERP may be less likely to have such an effect.

Inhibition of p53-dependent apoptosis by decreasing PERP levels or activity may be achieved by various approaches, for example, administration of antisense nucleic acids that enter target cells to inhibit transcription or translation of nucleic acids that encode PERP, or administration of chemical compounds that inhibit the transcription or translation of PERP, increase the degradation of PERP mRNA or protein, or otherwise block PERP activity (e.g., by disrupting the interaction of PERP with other proteins).

Cardiac Failure

Cardiac hypoxia, myocardial infarction, and congestive heart failure are known to stimulate cardiomyocyte apoptosis. Because cardiomyocytes do not regenerate within the adult heart, it is desirable to minimize, and ideally, to prevent, cardiomyocyte cell death in patients with these and other cardiac diseases and conditions. Several lines of evidence show that p53 is involved in cardiomyocyte apoptosis. For example, cardiomyocytes undergoing hypoxia- induced apoptosis display increased p53 transcriptional activating activity and protein accumulation, and overexpression of p53 in cardiomyocytes is sufficient to induce apoptosis (Long et al., J. Clin. Invest. 99:2635-2643, 1997). Moreover, cardiomyocytes undergoing apoptosis induced by chemical inhibition of the vacuolar proton ATPase display a marked increase in p53 mRNA accumulation (Long et al., J. Clin. Invest. 101: 1453-1461, 1998), and p53 binding to the bax promoter (a transcriptional target of p53 that stimulates cell death) is increased in apoptotic cardiomyocytes from failing hearts (Leri et al., Circulation 97: 194-203, 1998). Interruption of the p53-dependent or p53- independent cell death pathways by inhibiting PERP expression or activity should enhance cardiomyocyte survival and preserve cardiac function in patients that have or are at risk for cardiac diseases and injuries.

Neurode generation

p53 has been shown to be involved in neuronal apoptosis. For example, p53 overexpression induces neuronal death in vitro; conversely, neurons from p53 null mice (p53-/-) display higher resistance in vivo and in vitro to apoptosis induced by excitotoxicity and DNA-damaging agents

(Hughes et al., Neuroreport 8:v-xii, 1997). Increased p53 expression has been observed in Alzheimer's brains (de la Monte et al., J. Neurol. Sci. 152:73-83, 1997; Kitamura et al., Biochem. Biophys. Res. Commun. 232:418-421, 1997). Moreover, p53 appears to be involved in neuronal death in Parkinson's disease: p53 levels and activity have been observed to be up-regulated in cerebellar granule neurons undergoing dopamine toxicity-induced apoptosis (Daily et al., Cell. Mol. Neurobiol., 19:261-276, 1999) and in PC12 cells undergoing 6- hydroxydopamine toxicity-induced apoptosis (Blum et al., Brain Res. 751: 139- 142, 1997). Uberti et al. (Eur. J. Neurosci. 10:246-254, 1998) have shown that p53-specific antisense oligonucleotides inhibit glutamate excitotoxicity in rat cerebellar neurons. Moreover, Banasiak and Haddad (Brain Res. 797:295-304, 1998) have shown that p53-specific antisense oligonucleotides increase neuronal survival after exposure to hypoxic conditions (which occurs, e.g., during a stroke or other ischemic event). Our identification of PERP as a downstream target of p53 in the p53-dependent apoptosis pathway suggests that agents that inhibit PERP expression or activity are likely to be useful for increasing neuronal survival in patients that suffer from or are at risk for neurodegenerative diseases and injuries of the central and peripheral nervous system, e.g., but not limited to, stroke, Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis.

Retinal Degeneration

Apoptosis of photoreceptor cells has been demonstrated to occur in virtually all retinal pathologies, including retinitis pigmentosa, chemical toxicity, retinal detachment, glaucoma, diabetes, and axotomy. Ali et al. (Curr. Eye Res. 17:917-923, 1998) have shown that the absence of p53 delays retinal degeneration in a mouse model of inherited retinal degeneration, indicating that apoptotic cell death in the retinae of these mice is p53-dependent. In keeping with its position downstream of p53 in the p53-dependent cell death pathway, inhibition of PERP expression or activity is likely to provide significant therapeutic benefit in treating diseases involving retinal degeneration. Moreover, the accessibility of the eye makes it particularly amenable to gene therapy. Cutaneous lupus erythematosis

Cutaneous lupus erythematosus is an autoimmune disease involving excessive apoptosis of epidermal cells. Expression of p53 is higher than normal in epidermal cells from patients having this disease (Chung et al., Am. J. Dermatopathol., 20:233-241, 1998). Therefore, inhibition of the p53- dependent apoptotic pathway via inhibition of PERP may be useful in treating patients with cutaneous lupus erythematosis.

Inhibition of PERP expression or activity for treatment of other degenerative diseases or conditions

As described above, we observed that PERP expression is up- regulated in cells undergoing p53-dependent cell death. However, we noted that p53-deficient cells undergoing p53 -independent apoptosis also expressed PERP. Therefore, PERP may also be involved in p53-independent cell death. Accordingly, inhibition of PERP may constitute a novel therapeutic approach for ameliorating the effects of diseases that involve cell death pathways other than the p53-mediated cell death pathway. For example, inhibition of PERP may be a useful therapeutic approach for treating autoimmune diseases (e.g., autoimmune diabetes mellitus and acquired immunodeficiency syndrome (AIDS), degenerative diseases (e.g., Duchenne's muscular dystrophy), or for inhibiting rejection of transplanted organs, tissues, or cells. For example, prior to transplantation of dopaminergic neurons into a Parkinson's patient, or insulin-secreting pancreatic beta cells into a diabetes patient, the cells could be genetically modified ex vivo to express a PERP antisense nucleic acid. This may increase the survival of the transplanted cells and thus increase the efficacy of the transplant. Antisense Therapy

As described above, p53-dependent apoptosis occurs in numerous degenerative diseases and injuries. Therefore, inhibition of the p53 cell death pathway in patients having or at risk for such diseases should be useful for preventing or slowing the rate of disease progression. In addition to inhibiting p53 itself, the p53-dependent cell death pathway may be blocked by inhibiting PERP activity. Moreover, inhibition of PERP may also be useful for inhibiting p53-independent apoptosis.

Antisense therapy is based on the well-known principle of suppressing gene expression by intracellular hybridization of endogenous nucleic acid (genomic DNA or mRNA) molecules encoding a protein of interest with a complementary antisense nucleic acid, such as an antisense oligonucleotide or antisense RNA. Antisense nucleic acids may inhibit protein expression at the transcriptional level, at the translational level, or at both levels. Antisense oligonucleotides or antisense RNA, generated by well-known methods, may be administered to patients by conventional drug delivery techniques. The antisense nucleic acids enter the appropriate cell type and hybridize with the endogenous target nucleic acid to inhibit transcription or translation of the target protein. Antisense mRNA may also be provided intracellularly to a patient by administration of a gene therapy vector encoding an antisense RNA of interest. Expression of the antisense RNA may be limited to a particular cell type, for example, by placing a DNA molecule encoding the antisense RNA under the transcriptional regulation of a tissue-specific promoter. Inhibition of PERP transcription or translation using PERP antisense RNA increases a cell's resistance to various apoptotic stimuli. Numerous examples of therapeutic benefit derived from antisense therapy are known in the art. Just a few representative examples are described in: Gokhale et al., Gene Ther. 4: 1289-1299, 1997; Martens et al., Proc. Nad. Acad. Sci. USA 95:2664-2669, 1998; Offensperger et al., Mol. Biotechnol. 9:161-170, 1998; Kondo et al., Oncogene 16:3323-3330, 1998; and Higgens et al., Proc. Nad. Acad. Sci. USA 90:9901-9905, 1993.

PERP antisense nucleic acids contain at least 8 consecutive nucleotides that are complementary to a PERP mRNA or DNA sequence, and preferably contain at least 14-30 consecutive nucleotides that are complementary to a PERP mRNA or DNA. PERP antisense nucleic acids may contain at least, 40, 60, 85, 120, or more consecutive nucleotides that are complementary to a PERP mRNA or DNA, and may be as long as a full-length PERP gene or mRNA.

Any region of the human PERP coding or non-coding sequence may be used as a target for antisense inhibition of PERP transcription or translation, and particular sequences for PERP antisense nucleic acids may be selected by well-known approaches. For example, if desired, computer algorithms may be used to identify sequences that form the most stable hybridization duplexes. Computer algorithms may also be used to identify regions of the PERP sequence that are relatively accessible within a folded mRNA molecule; antisense nucleic acids against such regions are more likely to effectively inhibit translation of PERP mRNA. Computer algorithms that may be used to identify optimal PERP sequences for generating antisense nucleic acids include, but are not limited to, OLIGO 5.0 from National Biosciences Inc. (http://www.sxst.it/nbi olg.htm) and MFOLD

(http://mfold2.wustl.edu/~mfold/rna/forml.cgi). References describing algorithms for predicting secondary structure are described in M. Zuker et al. "Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide." in: RNA Biochemistry and Biotechnology, J. Barciszewski & B.F.C. Clark, eds., NATO ASI Series, Kluwer Academic Publishers (1999) and in Mathews et al. J. Mol. Biol. 288:911-940 (1999).

Therapy

Nucleic acids of the invention and compounds identified using any of the methods disclosed herein may be administered to patients or experimental animals with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients or experimental animals. Although intravenous administration is preferred, any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found in, for example, Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for molecules of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

Test Compounds

In general, novel drugs for modulation of PERP expression or activity (or mimicry of PERP activity) may be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available, e.g., from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA). In addition, natural and synthetically produced libraries are generated, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their apoptosis-modulatory activities should be employed whenever possible.

When a crude extract is found to modulate (i.e., stimulate or inhibit) or mimic PERP expression or activity, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having an activity that stimulates or inhibits PERP expression or activity (or mimics the same). The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value may be subsequently analyzed using animal models for diseases in which it is desirable to decrease PERP expression or activity (for example, to decrease levels in cells that are susceptible to apoptosis, such as cardiomyocytes in an animal prone to myocardial infarctions), or to increase PERP expression or activity or to mimic its activity (for example, in cancer cells in an animal tumor model, thereby rendering the tumor cells more susceptible to apoptosis).

Below are examples of high-throughput systems useful for evaluating the efficacy of a molecule or compound for increasing or decreasing PERP expression or activity, or for mimicking its activity.

Assays for identifying compounds that modulate apoptotic cell death

We have shown that expression of PERP is up-regulated in cells undergoing apoptotic death. Therefore, measurements of PERP levels may be used to determine the apoptotic status of cells in a sample. Such measurements may be employed in high-throughput screens for the identification of novel therapeutic compounds that modulate (i.e., stimulate or inhibit) apoptotic death of various types of cells under various physiological conditions.

For example, to identify a novel compound that stimulates apoptosis in cells lacking (or having decreased levels of) functional p53 (such as many types of tumor cells), tumor cells or growth-deregulated cells that lack functional p53 (such as EIA p53-/- MEFs) may be treated with various test compounds, after which PERP mRNA or protein levels may be measured using well-known approaches, such as RT-PCR (for mRNA) or ELISA (for protein). An increase in PERP expression indicates a pro-apoptotic compound that may then be further tested using appropriate cell culture and animal models for its usefulness as an anti-cancer agent. Conversely, to identify a novel compound that inhibits apoptosis, cultured cells that are known to undergo apoptosis when exposed to an appropriate pro-apoptotic stimulus are exposed to test compounds either before, after, or concurrent with exposure to the apoptotic stimulus. A decrease in PERP mRNA or protein levels relative to a control apoptotic sample not treated with the compound indicates an anti-apoptotic compound that may then be further tested for its usefulness in treating diseases or conditions that involve excessive, pathological apoptosis, e.g. (but not limited to), neurodegenerative diseases, retinal degenerative diseases, cardiac degenerative diseases, and transplant rejection.

Various cell culture models of apoptosis are known in the art; any of these may be used to identify anti-apoptotic compounds with potential therapeutic utility. For example, cultured neurons and cardiomyocytes undergo apoptosis when subjected to hypoxic conditions, neurons undergo apoptosis when exposed to high concentrations of glutamine, NMD A, or other neuroexcitatory compounds, and cultured fibroblasts, and many other types of cultured cells undergo apoptosis after serum or growth factor withdrawal, staurosporine exposure, DNA damage, or exposure to reactive oxygen species. One of ordinary skill in the art may readily identify which of the known cell culture models would be appropriate for the high-throughput screens of the invention.

In addition, apoptosis may be induced by expressing vector-encoded PERP within the experimental cells. PERP expression may be placed under one of the many known regulatable promoters, such as a promoter that becomes transcriptionally active in the presence of a hormone (e.g., a steroid hormone such as estrogen), an antibiotic (such as tetracycline), metal ions (e.g., zinc), heat shock, or hypoxic conditions. An inducible promoter allows the generation of stable cell lines that undergo PERP-mediated apoptosis when PERP is inducibly expressed. Such cells may be used in high-throughput screens for identification of compounds that involved PERP-mediated cell death, which may be monitored by any of the many apoptosis detection assays known in the art or disclosed herein.

ELISA for the detection of compounds that modulate apoptotic cell death

Enzyme-linked immunosorbant assays (ELISAs) are easily incorporated into high-throughput screens designed to test large numbers of compounds for their ability to modulate levels of a given protein. When used in the methods of the invention, changes in the level of PERP protein in a sample, relative to a control, reflect changes in the apoptotic status of the cells within the sample. Protocols for ELISA may be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998. Lysates from cells treated with test compounds are prepared (see, for example, Ausubel et al., supra), and are loaded into the wells of microtiter plates coated with "capture" antibodies against PERP. Unbound antigen is washed out, and a PERP-specific antibody, coupled to an agent to allow for detection, is added. Agents allowing detection include alkaline phosphatase (which can be detected following addition of colorimetric substrates such as p- nitrophenolphosphate), horseradish peroxidase (which can be detected by chemiluminescent substrates such as ECL, commercially available from Amersham) or fluorescent compounds, such as FITC (which can be detected by fluorescence polarization or time-resolved fluorescence). The amount of antibody binding, and hence the level of PERP protein within a lysate sample, is easily quantitated on a microtiter plate reader. An increase in the level of PERP in a treated sample, relative to the level of PERP in an untreated sample, indicates a compound that stimulates apoptosis. Conversely, a decrease in the level of PERP in a treated sample, relative to the level of PERP in an untreated sample, indicates a compound that inhibits apoptosis.

Any person having ordinary skill in the art will understand that the appropriate controls should be included in each assay. The skilled artisan will know which controls to include, depending upon whether a pro-apoptotic compound or an anti-apoptotic compound is being sought, and depending upon the particular cell culture model being used for the assay.

Quantitative PCR of PERP mRNA as an assay for compounds that modulate apoptotic cell death

The polymerase chain reaction (PCR), when coupled to a preceding reverse transcription step (RT-PCR), is a commonly used method for detecting vanishingly small quantities of a target mRNA. When performed within the linear range, with an appropriate internal control target (employing, for example, a housekeeping gene such as β-actin or GAPDH), such quantitative PCR provides an extremely precise and sensitive means of detecting slight modulations in mRNA levels. Moreover, this assay is easily performed in a 96- well format, and hence is easily incorporated into a high-throughput screening assay. The appropriate cells (depending upon whether the screen is for pro- apoptotic compounds or anti-apoptotic compounds) are cultured, treated with test compounds, and (if screening for anti-apoptotic compounds) exposed to an appropriate apoptotic stimulus. The cells are then lysed, the mRNA is reverse- transcribed, and the PCR is performed according to commonly used methods (such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998), using oligonucleotide primers that specifically hybridize with PERP mRNA. Changes in the levels of PERP RT-PCR product from samples exposed to test compounds, relative to control samples, indicate test compounds with apoptosis-modulating activity, i.e., an increase in the level of PERP RT-PCR product indicates a compound that stimulates apoptosis, and, conversely, a decrease in the level of PERP RT- PCR product indicates a compound that inhibits apoptosis.

Primer sequences for PERP-specific RT-PCR amplification may be selected using any one of the many known primer selection programs, e.g., Primer3 (http://www-genome.wi.mit.edu/cgi-bin primer/primer3_www.cgi), or by other commonly-known approaches for selecting PCR primers.

Reporter gene assays for compounds that modulate apoptotic cell death

Assays employing the detection of reporter gene products are extremely sensitive and readily amenable to automation, hence making them ideal for the design of high-throughput screens. Assays for reporter genes may employ, for example, colorimetric, chemiluminescent, or fluorometric detection of reporter gene products. Many varieties of plasmid and viral vectors containing reporter gene cassettes are easily obtained. Such vectors contain cassettes encoding reporter genes such as lacZ/β-galactosidase, green fluorescent protein, and luciferase, among others. A genomic DNA fragment carrying a PERP-specific transcriptional control region (e.g., a promoter and/or enhancer) is first cloned using standard approaches (such as those described by Ausubel et al., supra). The DNA carrying the PERP transcriptional control region is then inserted, by DNA subcloning, into a reporter vector, thereby placing a vector-encoded reporter gene under the control of the PERP transcriptional control region. The activity of the PERP transcriptional control region operably linked to the reporter gene can then be directly observed and quantitated as a function of reporter gene activity in a reporter gene assay.

In one embodiment, for example, the PERP transcriptional control region could be cloned upstream from a luciferase reporter gene within a reporter vector. This could be introduced into the test cells, along with an internal control reporter vector (e.g., a lacZ gene under the transcriptional regulation of the β-actin promoter). After the cells are exposed to the test compounds and apoptotic stimulus (if testing for anti-apoptotic compounds), reporter gene activity is measured and PERP reporter gene activity is normalized to internal control reporter gene activity. An increase in PERP reporter gene activity indicates a compound that stimulates apoptosis and a decrease in PERP reporter gene activity indicates a compound that inhibits apoptosis.

Interaction trap assays

Examination of the PERP primary amino acid sequence has revealed an amino acid motif (AWGRAAAATLF; SEQ ID NO: 11) that bears sequence similarity to a motif known as Bcl-2 Homology Region 1 (BH1). This peptide motif is known to mediate protein-protein interactions (for example, between Bcl-2 and Bax) and is present in several known pro-apoptotic and anti- apoptotic proteins. Examples of such proteins having BH1 domains include Bcl-2 (NWGRIVAFFEFG; SEQ ID NO: 12), Bcl-w (NWGRLVAFFVFG; SEQ ID NO: 13), Bcl-x (NWGRIVAFFSFG; SEQ ID NO: 14), and Bax (NWGRVVALFYFA; SEQ ID NO: 15). Although not wishing to be bound by theory, it is possible that PERP exerts its pro-apoptotic effects by interacting with other proteins via its BH1 domain or via other domains within the protein.

Two-hybrid methods, and modifications thereof, may be used to identify novel proteins that interact with PERP, and hence may be naturally occurring regulators of PERP. Such assays also may be used to screen for compounds that modulate the physical interactions of PERP with itself or with other proteins. Regulators of PERP, e.g., proteins that interfere with or enhance the interaction between PERP and other proteins may identified by the use of a three-hybrid system. Such assays are well-known to skilled artisans, and may be found, for example, in Ausubel et al., supra.

Synthesis of PERP polypeptides

Nucleic acids that encode PERP or fragments thereof may be introduced into various cell types or cell-free systems for expression of PERP polypeptides and fragments, thereby allowing purification of PERP for biochemical characterization, large-scale production, antibody production, and patient therapy.

Eukaryotic and prokaryotic PERP expression systems may be generated in which PERP gene sequences are introduced into a plasmid or other vector, which is then used to transform living cells. Constructs in which the PERP cDNAs containing the entire open reading frames inserted in the correct orientation into an expression plasmid may be used for protein expression. Alternatively, portions of the PERP gene sequences, including wild-type or mutant PERP sequences, may be inserted. Prokaryotic and eukaryotic expression systems allow various important functional domains of the PERP proteins to be recovered, if desired, as fusion proteins, and then used for binding, structural, and functional studies and also for the generation of appropriate antibodies. Since PERP protein expression induces apoptosis in at least some types of cells, it may be desirable to express the protein under the control of an inducible promoter.

Typical expression vectors contain promoters that direct the synthesis of large amounts of mRNA corresponding to the inserted PERP nucleic acid in the plasmid-bearing cells. They may also include eukaryotic or prokaryotic origin of replication sequences allowing for their autonomous replication within the host organism, sequences that encode genetic traits that allow vector- containing cells to be selected for in the presence of otherwise toxic drugs, and sequences that increase the efficiency with which the synthesized mRNA is translated. Stable long-term vectors may be maintained as freely replicating entities by using regulatory elements of, for example, viruses (e.g., the OriP sequences from the Epstein Ban Virus genome). Cell lines may also be produced that have integrated the vector into the genomic DNA, and in this manner the gene product is produced on a continuous basis.

Expression of foreign sequences in bacteria, such as Escherichia coli requires the insertion of the PERP nucleic acid sequence into a bacterial expression vector. Such plasmid vectors contain several elements required for the propagation of the plasmid in bacteria, and for expression of the DNA inserted into the plasmid. Propagation of only plasmid-bearing bacteria is achieved by introducing, into the plasmid, selectable marker-encoding sequences that allow plasmid-bearing bacteria to grow in the presence of otherwise toxic drugs. The plasmid also contains a transcriptional promoter capable of producing large amounts of mRNA from the cloned gene. Such promoters may be (but are not necessarily) inducible promoters that initiate transcription upon induction. The plasmid also preferably contains a polylinker to simplify insertion of the gene in the correct orientation within the vector. Once the appropriate expression vectors containing a PERP gene, or fragment, fusion, or mutant thereof, are constructed, they are introduced into an appropriate host cell by transformation techniques, such as, but not limited to, calcium phosphate transfection, DEAE-dextran transfection, electroporation, microinjection, protoplast fusion, or liposome-mediated transfection. The host cells that are transfected with the vectors of this invention may include (but are not limited to) E. coli or other bacteria, yeast, fungi, insect cells (using, for example, baculoviral vectors for expression), or cells derived from mice, humans, or other animals. Mammalian cells can also be used to express the PERP protein using a vaccinia virus expression system. In vitro expression of PERP proteins, fusions, polypeptide fragments, or mutants encoded by cloned DNA may also be used. Those skilled in the art of molecular biology will understand that a wide variety of expression systems and purification systems may be used to produce recombinant PERP polypeptides and fragments thereof. Some of these systems are described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998.

Once a recombinant protein is expressed, it can be isolated from cell lysates using protein purification techniques such as affinity chromatography (see, e.g., Ausubel et al., supra). Once isolated, the recombinant protein can, if desired, be purified further by e.g., by high performance liquid chromatography (HPLC; e.g., see Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, Eds., Elsevier, 1980).

Polypeptides of the invention, particularly short PERP fragments and longer fragments of the N-terminus and C-terminus of the PERP protein, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, IL). PERP Antibodies

In order to prepare polyclonal antibodies, PERP proteins, fragments of PERP proteins, or fusion proteins containing defined portions of PERP proteins may be synthesized in bacteria by expression of corresponding DNA sequences in a suitable cloning vehicle. The proteins can be purified, and then coupled to a carrier protein and mixed with Freund's adjuvant (to enhance stimulation of the antigenic response in an innoculated animal) and injected into rabbits or other laboratory animals. Alternatively, protein can be isolated from PERP- expressing cultured cells. Following booster injections at bi-weekly intervals, the rabbits or other laboratory animals are then bled and the sera isolated. The sera can be used directly or can be purified prior to use by various methods, including affinity chromatography employing reagents such as Protein A- Sepharose, antigen-Sepharose, and anti-mouse-Ig-Sepharose. The sera can then be used to probe protein extracts from PERP-expressing tissue electrophoretically fractionated on a polyacrylamide gel to identify PERP proteins. Alternatively, synthetic peptides can be made that correspond to the antigenic portions of the protein and used to innoculate the animals. See, e.g., Ausubel et al., supra.

Alternatively, monoclonal antibodies may be prepared using the PERP proteins described above and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York, NY, 1981 ; Ausubel et al., supra). Once produced, monoclonal antibodies are also tested for specific PERP protein recognition by Western blot or immunoprecipitation analysis (by the methods described in Ausubel et al., supra). Antibodies of the invention may be produced using PERP amino acid sequences that do not reside within highly conserved regions, and that appear likely to be antigenic, as analyzed by criteria such as those provided by the Peptide Structure Program (Genetics Computer Group Sequence Analysis Package, Program Manual for the GCG Package, Version 7, 1991) using the algorithm of Jameson and Wolf (CABIOS 4: 181, 1988). These fragments can be generated by standard techniques (Ausubel et al., supra). GST fusion proteins are expressed in E. coli and purified using a glutathione-agarose affinity matrix as described in Ausubel et al., supra). To generate rabbit polyclonal antibodies, and to minimize the potential for obtaining antisera that is non-specific, or exhibits low-affinity binding to a PERP, two or three fusions are generated for each protein, and each fusion is injected into at least two rabbits. Antisera are raised by injections in series, preferably including at least three booster injections.

All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations following, in general, the principles of the invention and including such departures from the present disclosure within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

What is claimed is:

Claims

1. A substantially pure PERP polypeptide.

2. The PERP polypeptide of claim 1, wherein said polypeptide is a mammalian polypeptide.

3. The PERP polypeptide of claim 2, wherein said polypeptide is a mouse or human polypeptide.

4. The PERP polypeptide of claim 3, wherein said polypeptide comprises an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 2.

5. The PERP polypeptide of claim 4, wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

6. A substantially pure nucleic acid, wherein said nucleic acid comprises a nucleotide sequence that encodes a PERP polypeptide.

7. The substantially pure nucleic acid of claim 6, wherein said nucleic acid comprises a nucleotide sequence that encodes a mammalian PERP polypeptide.

8. The substantially pure nucleic acid of claim 7, wherein said nucleic acid comprises a nucleotide sequence that encodes a mouse or human PERP polypeptide.

9. The substantially pure nucleic acid of claim 8, wherein said nucleic acid comprises a nucleotide sequence that encodes a PERP polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 2.

10. The substantially pure nucleic acid of claim 9, wherein said nucleic acid comprises a nucleotide sequence that encodes a PERP polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

11. The substantially pure nucleic acid of claim 10, wherein said nucleic acid comprises the PERP coding sequence of SEQ ID NO: 1.

12. The substantially pure nucleic acid of claim 11, wherein said nucleic acid is within an expression vector, wherein said nucleic acid is operably linked to a promoter.

13. A non-human transgenic animal, wherein said animal comprises a substantially pure nucleic acid comprising a nucleotide sequence that encodes a PERP polypeptide.

14. The non-human transgenic animal of claim 13, wherein said animal is a mouse.

15. A non-human animal, wherein one or both endogenous alleles encoding PERP are mutated, disrupted, or deleted in said non-human animal.

16. The non-human animal of claim 15, wherein said non-human animal is a mouse, pig, goat, sheep, or cow.

17. A substantially pure nucleic acid that comprises at least 14 consecutive nucleotides that display at least 85%, 90%, 92%, 95%, or 98% sequence identity to a nucleotide sequence that is complementary to a PERP nucleic acid.

18. The substantially pure nucleic acid of claim 17, wherein said nucleic acid comprises at least 16, 18, 22, 25, 50, 75, or 100 consecutive nucleotides that display at least 85%, 90%, 92%, 95%, or 98% sequence identity to a nucleotide sequence that is complementary to a PERP nucleic acid.

19. The nucleic acid of claim 17, wherein said nucleic acid hybridizes under high stringency conditions to a PERP nucleic acid.

20. A substantially pure nucleic acid comprising at least 14 nucleotides, wherein said nucleic acid hybridizes under high stringency conditions to a PERP nucleic acid.

21. The substantially pure nucleic acid of claim 20, wherein said substantially pure nucleic acid comprises at least 16, 18, 22, 25, 50, 75, or 100 nucleotides.

22. The substantially pure nucleic acid of claim 17 or 20, wherein said substantially pure nucleic acid is an antisense nucleic acid.

23. A method of detecting the presence of a PERP nucleic acid, said method comprising:

(a) providing a sample;

(b) contacting said sample, under high stringency conditions, with a nucleic acid probe, wherein said probe hybridizes under high stringency conditions to a PERP nucleic acid; and

(c) assaying for the presence of hybridized probe, wherein the presence of said hybridized probe indicates the presence of a PERP nucleic acid.

24. The method of claim 23, wherein said method is for detecting PERP biological activity.

25. The method of claim 23, wherein said PERP nucleic acid is genomic DNA, cDNA, or mRNA.

26. The method of claim 23, wherein said method further comprises use of the polymerase chain reaction (PCR), wherein the presence of a PCR product indicates the presence of said PERP nucleic acid.

27. A method of inhibiting expression of PERP in a cell, said method comprising introducing into said cell a PERP antisense nucleic acid.

28. A method for stimulating apoptosis in a population of cells, said method comprising introducing into said cells substantially pure PERP polypeptide, wherein said PERP polypeptide stimulates apoptosis in said cells, compared to cells not containing said PERP polypeptide.

29. The method of claim 28, wherein said PERP polypeptide is encoded by a substantially pure PERP nucleic acid, wherein said nucleic acid is introduced into said cells.

30. The method of claim 27 or 28, wherein said cells are tumor cells.

31. The method of claim 27 or 28, wherein said cells are exposed to an apoptotic stimulus before or after said PERP polypeptide is introduced into said cells.

32. The method of claim 31, wherein said apoptotic stimulus is gamma inadiation or a chemotherapeutic agent.

33. A method for inhibiting apoptosis in a population of cells having an increased risk for undergoing apoptosis, said method comprising introducing into said cells a PERP antisense nucleic acid, wherein said PERP antisense nucleic acid decreases the level of PERP in said cells, wherein said decrease inhibits apoptosis in said cells.

34. The method of claim 33, wherein said increased risk for undergoing apoptosis is caused by: exposure to gamma inadiation, exposure to a chemotherapeutic agent, exposure to a toxin, exposure to hypoxia, an injury, a degenerative disease, or an attack by cells of the immune system.

35. A method of identifying a compound that modulates apoptosis, said method comprising the steps of:

(a) exposing a sample to a test compound, wherein said sample comprises a PERP nucleic acid, a PERP reporter gene, or a PERP polypeptide; and

(b) assaying for a change in the level of PERP biological activity in said sample, relative to a sample not exposed to said test compound, wherein an increase in said level of PERP biological activity in said sample indicates a compound that stimulates apoptosis, and a decrease in said level of PERP biological activity in said sample indicates a compound that inhibits apoptosis.

36. The method of claim 35, wherein said PERP nucleic acid is genomic DNA, cDNA, mRNA, cRNA, or a substantially pure genomic DNA fragment.

37. The method of claim 35, wherein said PERP nucleic acid, PERP reporter gene, or PERP polypeptide is within a cell, wherein said cell is exposed to said test compound.

38. An antibody that specifically binds PERP.

39. The antibody of claim 38, wherein said antibody is a monoclonal antibody or a polyclonal antibody.

40. A method of detecting the presence of a PERP polypeptide, said method comprising:

(a) contacting a sample with an antibody that specifically binds said PERP polypeptide; and

(b) assaying for the binding of said antibody to said PERP polypeptide, wherein said binding of said antibody to said PERP polypeptide indicates the presence of a PERP polypeptide.

41. The method of claim 40, wherein said method is for detecting PERP biological activity.

42. A substantially pure nucleic acid that comprises at least 14 consecutive nucleotides that display at least 85%, 90%, 92%, 95%, or 98% sequence identity to a nucleotide sequence within a PERP nucleic acid, wherein said substantially pure nucleic acid is not an expressed sequence tag of SEQ ID NOS: 16-259.