WO2001020335A2 - Methods for modulating cell proliferation - Google Patents

Abstract

The invention features methods for modulating cell proliferation by modulating the interaction between NRAGE and E2F proteins. The invention also features methods of identifying compounds that modulate this interaction.

Description

METHODS FOR MODULATING CELL PROLIFERATION

Background of the Invention The invention relates to methods for regulating cell growth and survival. The regulation of cell growth and survival is believed to be under the control of a wide variety of signaling cascades. One such cascade is that which transduces the presence of a neurotrophin ligand. Early in vivo and in vitro experiments demonstrated that nerve growth factor (NGF) plays critical roles in the development of the nervous system. The cloning of brain-derived neurotrophic factor (BDNF) subsequently revealed a homology with NGF that spurred the cloning and characterization of neurotrophin- 3 (NT-3), neurotrophin- 4/5 (NT-4/5) and neurotrophin-6 (NT-6). Each of these proteins promotes survival of specific populations of neurons and affects aspects of the neuronal phenotype.

The mammalian neurotrophins interact with two types of cell surface receptors. The trk receptors (e.g., trkA, trkB, and trkC) are highly-related transmembrane receptor tyrosine kinases, each of which preferentially binds one or a subset of the neurotrophin family members, trk receptors play a critical role in mediating the effects of the neurotrophins, and their activation results in the effects typically associated with neurotrophin action. The p75NTR receptor (p75NTR), which binds all neurotrophins with approximately equal affinity, is a member of the tumor necrosis factor (TNF) receptor superfamily. In contrast to the rapid progress made in elucidating the mechanism of action of the trk receptors, the physiological roles of the p75NTR have been more difficult to discern. At present, the actions of p75NTR fall into two categories. First, p75NTR appears to functionally collaborate with trk receptors to either enhance or reduce neurotrophin-mediated trk receptor activation. Second, p75NTR acts autonomously to activate signaling cascades that may be involved in apoptosis and inflammation. A number of proteins which directly interact with the intracellular domains of members of the TNF receptor superfamily have been identified, including TRADD, FADD, and members of the TRAF family. Generally, the intracellular domain of p75NTR is highly conserved across species, but not conserved with other members of the TNF receptor superfamily. The exception to the general lack of homology between p75NTR and the TNF receptor superfamily is the presence of a 90 amino acid stretch termed the "death domain." In TNF receptor- 1 (TNFR-1), fas, DR3, and other related receptors, the death domain is required to mediate interactions with either FADD or TRADD. We have previously found, however, that the p75NTR does not bind

TRADD or FADD proteins. The failure of p75NTR to bind FADD or TRADD is likely due to the fact that the tertiary structure of the p75NTR death domain differs considerably from these other receptors, suggesting that p75NTR must facilitate apoptosis through another mechanism. Thus, the mechanism by which neurotrophins signal via p75NTR to modulate cell proliferation and apoptosis remains unknown.

A second signal transduction cascade critical for the regulation of cell growth and survival is the retinoblastoma/E2F cascade. Current models of cell cycle progression suggest that the D- and E-type cyclins, as well as cdk 4/6 and cdk2, their cyclin-dependent kinase counterparts, are key regulators of the Gl to S phase transition. These molecules are synthesized as part of the early response to mitogemc stimulation and together act to phosphorylate the retinoblastoma protein (pRB) and related family members. The phosphorylation of pRB at the mid-Gl restriction point releases the E2F transcription factors and allows them to activate genes crucial for cell cycle progression; E2F-mediated transcription is thought to irreversibly commit cells to progress into S phase after which they are competent only to complete the cell cycle or undergo apoptosis. Unphosphorylated pRB and related proteins (e.g., pl07 and pl30) remain bound to the E2F family of transcription factors and restrict cell-cycle related gene expression through sequestration or direct trancriptional repression. There is a need for drugs that are useful for the treatment of human diseases in which there is inappropriate cell proliferation (e.g., cancer) or cell death (e.g., neurodegeneration, muscular dystrophy). Understanding the molecular mechanisms by which neurotrophins function will reveal targets for pharmacological intervention, as well as pharmaceuticals themselves, that could be of substantial benefit in treating these diseases.

Summary of the Invention We have discovered evidence of a signal transduction pathway that links neurotrophin signaling and regulation of transcriptional activity of E2F proteins. Specifically, we have found that NRAGE (a member of the MAGE family of proteins), is part of the neurotrophin-E2F signal transduction pathway, binds to both p75NTR and E2F, and is capable of regulating a cell's progression through the cell cycle. We also discovered that NRAGE forms complexes with itself and other MAGE proteins. Accordingly, in a first aspect, the invention features a method for modulating cell proliferation. The method includes administering an NRAGE polypeptide to the cell.

In a second aspect, the invention features another method for modulating cell proliferation. This method includes administering to the cell a compound that modulates an NRAGE biological activity.

In a preferred embodiment of the second aspect, the compound is selected from the group consisting of a chemical, a drug, an NRAGE antisense nucleic acid molecule, and an antibody that specifically binds to an NRAGE polypeptide. A preferred antibody is a neutralizing antibody. In another preferred embodiment, the biological activity is binding to an

E2F protein.

In a preferred embodiment of the first or second aspect, the cell is in a mammal (e.g., a human or a rodent). Preferably, the modulating of cell proliferation is decreasing the proliferation in the mammal diagnosed as having a cell proliferation disease.

In a third aspect, the invention features a method for identifying a compound that modulates cell proliferation. The method includes (a) providing a cell expressing an NRAGE polypeptideand an E2F polypeptide; (b) contacting the cell with a candidate compound; and (c) monitoring the level of binding of the NRAGE polypeptide to the E2F polypeptide, wherein a change in the level of binding in response to the candidate compound relative to a level of binding in a cell not contacted with the candidate compound identifies the candidate compound as a compound that modulates cell proliferation. Preferred E2F polypeptides are mammalian E2F-1, E2F-2, E2F-3, E2F-4, and E2F-5.

In a fourth aspect, the invention features a method for diagnosing a mammal for the presence of a cell proliferation disease or an increased likelihood of developing the disease. The method includes measuring the level of binding between an NRAGE polypeptide and an E2F polypeptide, wherein an alteration in the level of binding, relative to a level of binding in a sample from an unaffected mammal, indicates that the mammal has the disease or increased likelihood of developing the disease. In preferred aspects of the third or aspect, the cell is from a mammal (e.g., a human or a rodent) and the E2F polypeptide is mammalian E2F- 1 , E2F-2, E2F-3 , E2F-4, or E2F-5.

An "NRAGE biological activity" includes, but is not limited to: binding of NRAGE to at least one E2F protein; NRAGE-mediated degradation of at least one E2F protein; and NRAGE-mediated modulation of cell proliferation. By "polypeptide" is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.

NRAGE polypeptides that are a part of the invention include those polypeptides that bind to E2F proteins, are capable of binding an antibody which specifically binds NRAGE, or have an NRAGE biological activity in a cell. Preferred NRAGE polypeptides are those encoded by the amino acid sequence of SEQ ID NO: 1 (Fig. 1A) or SEQ ID NO: 2 (Fig. IB).

NRAGE nucleic acids that are a part of the invention include those encoding polypeptides that bind to an E2F protein, are capable of binding an antibody which specifically binds NRAGE, or have an NRAGE biological activity in a cell. Preferred NRAGE nucleic acids are those having the nucleotide sequence of SEQ ID NO: 3 (Fig. 1C) or SEQ ID NO: 4 (Fig. ID).

By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.

Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By "high stringency conditions" is meant hybridization in 2X SSC at 40°C with a DNA probe length of at least 40 nucleotides. For other definitions of high stringency conditions, see F. Ausubel et al., Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6, John Wiley & Sons, New York, NY, 1994, hereby incorporated by reference. By "substantially pure polypeptide" is meant a polypeptide 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 an NRAGE polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure NRAGE polypeptide may be obtained, for example, by extraction from a natural source (e.g. a neuronal cell), by expression of a recombinant nucleic acid encoding an NRAGE polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A polypeptide is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a polypeptide which 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 include those which naturally occur in eukaryotic organisms but are synthesized in E. coli or other prokaryotes. By "substantially pure nucleic acid" is meant nucleic acid that is free of the genes which, in the natnrally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the nucleic acid. The term therefore includes, for example, a recombinant nucleic acid that is incorporated into a vector; into an autonomously replicating plasmid or virus; into the genomic nucleic acid of a prokaryote or a eukaryote cell; or that 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 nucleic acid that is part of a hybrid gene encoding additional polypeptide sequence. By "antisense," as used herein in reference to nucleic acids, is meant a nucleic acid sequence that is complementary to the coding strand of a gene, preferably, an NRAGE gene. Preferably the antisense nucleic acid molecule decreases the amount of transcription from the gene; more preferably, the decrease is at least 10%, and most preferably, the decrease is at least 50% when administered at the maximally effective dose.

By "substantially pure antibody" is meant antibody which is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques.

By "specifically binds" is meant an antibody that recognizes and binds to, for example, a human, mouse, or rat NRAGE polypeptide but does not substantially recognize and bind to other non-NRAGE molecules in a sample, e.g., a biological sample, that naturally includes protein. A preferred antibody binds to an NRAGE polypeptide sequence of Fig. 1A.

By "neutralizing antibodies" is meant antibodies that interfere with any of the biological activities of an NRAGE polypeptide, for example, the ability of NRAGE to bind to an E2F protein. The neutralizing antibody may reduce the ability of an NRAGE to bind to an E2F protein by 50%, more preferably by 70%, and most preferably by 90% or more. Any standard assay for the biological activity of NRAGE, including those described herein, may be used to assess potentially neutralizing antibodies that are specific for NRAGE.

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 candidate compound. By "treat" is meant to submit or subject an animal (e.g. a human), cell, lysate or extract derived from a cell, or molecule derived from a cell to a candidate compound.

By "candidate compound" is meant a chemical, be it naturally-occurring or artificially-derived, that is assayed for its ability to modulate an alteration in reporter gene activity or protein levels, by employing one of the assay methods described herein. Test compounds may include, for example, peptides, polypeptides, synthesized 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 or cells 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 "modulating" is meant changing, either by decrease or increase, in biological activity.

By "a decrease" is meant a lowering in the level of biological activity, as measured by a lowering/increasing of: a) protein, as measured by ELISA; b) reporter gene activity, of at least 30%, as measured by reporter gene assay, for example, /αcZ/β-galactosidase, green fluorescent protein, luciferase, etc.; c) mRNA, levels of at least 30%, as measured by PCR relative to an internal control, for example, a "housekeeping" gene product such as β-actin or glyceraldehyde 3 -phosphate dehydrogenase (GAPDH). In all cases, the lowering is preferably by 30%, more preferably by 40%, and even more preferably by 70%. By "an increase" is meant a rise in the level of biological activity, as measured by a lowering/increasing of: a) protein, measured by ELISA; b) reporter gene activity, as measured by reporter gene assay, for example, /αcZ/β- galactosidase, green fluorescent protein, luciferase, etc.; c) mRNA, as measured by PCR relative to an internal control, for example, a "housekeeping" gene product such as β-actin or glyceraldehyde 3 -phosphate dehydrogenase (GAPDH). Preferably, the increase is by 5% or more, more preferably by 15% or more, even more preferably by 2-fold, and most preferably by at least 3 -fold.

By "promoter" is meant a minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific, temporal-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' or intron sequence regions of the native gene. By "operably linked" is meant that a gene and one or more regulatory sequences 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 "pharmaceutically acceptable carrier" is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline solution. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington: The Science and Practice of Pharmacy. (19^th edition), ed. A. Gennaro, 1995, Mack PubUshing Company, Easton, PA.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Detailed Description of the Drawings Fig. 1A shows the polypeptide sequence for human NRAGE (SEQ ID NO:

1).

Fig. IB shows the polypeptide sequence for rat NRAGE (SEQ ID NO: 2). Fig. 1C shows the nucleic acid sequence for human NRAGE (SEQ LD

NO: 3).

Fig. ID shows the nucleic acid sequence for rat NRAGE (SEQ ID NO: 4).

Fig. 2 is a schematic illustration showing structural relationships among NRAGE and other MAGE family members. The black bars indicate sequences against which antibodies have been produced.

Fig. 3 is a photograph of a Western blot showing developmental and tissue expression of NRAGE. Lysates of tissues from E18 embryos and adult (Ad) rats were immunoblotted using a polyclonal antibody against NRAGE.

Fig. 4 is a schematic illustration showing the interactions of NRAGE with p75NTR and E2F family members. Black bars indicate putative interaction domains within each protein, rdd indicates "rapid degradation domain."

Fig. 5 is a series of photographs showing that NRAGE binds to E2F proteins in vitro. GST or an NRAGE-GST fusion protein bound to glutathione beads were incubated with E2F-2 or E2F-3 produced by in vitro translation. Pellets were then washed repeatedly and proteins released by incubation in sample buffer.

Fig. 6 is a photograph of a Western blot showing that tagged NRAGE is excluded from the nucleus. Cells were lysed in detergent-free buffer and disrupted in a dounce homogenizer. Crude cellular compartments were obtained by differential centrifugation and analyzed by immunoblot using NRAGE- specifϊc antibodies.

Fig. 7 is a series of photographs showing that NRAGE overexpression reduces E2F levels. 293 T cells were transfected with HA-tagged E2F-2 in the absence and presence of myc-tagged NRAGE. Forty-eight hours after transfection, cells were lysed, analyzed for protein content, and then analyzed by immunoblots.

Fig. 8 is a photograph showing that NRAGE is degraded through the proteasome. PC12nnr5 cells were incubated in the presence or absence of 50 uM lactacystin (Lcyn), a highly-specific proteasome inhibitor, for 4 hours. Cells were then lysed, analyzed for protein content, and then analyzed for endogenous NRAGE content by immunoblot.

Fig. 9 is a photograph showing that NRAGE binds to p75NTR in vitro. GST or p75NTR-GST fusion proteins bound to glutathione beads were incubated with NRAGE produced by in vitro translation. Top panel shows NRAGE specifically associating with p75NTR-GST but not with GST. Bottom panel shows that equivalent amounts of GST protein were used.

Fig. 10 is a photograph showing that overexpressed NRAGE and ρ75NTR interact in vitro. 293T cells were transfected with myc-NRAGE in the presence and absence of p75NTR. Cells were lysed and immunoprecipitated with a p75NTR monoclonal antibody.

Figs. 11A - 11C are a series of photographs showing that overexpression of NRAGE results in cell cycle arrest. Transfected Cos7 cells expressing a GFP-NRAGE fusion protein were incubated with BrdU for 1 hour, then fixed for immunocytochemistry. GFP and BrdU were detected using a specific antibodies. Cell nuclei were labeled with Hoescht stain.

Detailed Description of the Invention We have discovered evidence of a signal transduction pathway that links neurotrophin signaling and regulation of transcriptional activity of E2F proteins. Specifically, we have found that NRAGE (a member of the MAGE family of proteins), is part of the neurofrophin-E2F signal transduction pathway, binds to both p75NTR and E2F, and is capable of regulating a cell's progression through the cell cycle. We also discovered that NRAGE forms complexes with itself and other MAGE proteins. The foregoing discoveries provide new methods for regulating cell growth and survival. Additionally, the discovery allows for generation of drugs useful in the treatment of human diseases.

Identification of molecules that modulate NRAGE biological activity The effect of candidate molecules on NRAGE-mediated regulation of cell growth or survival may be measured at the level of translation by using the general approach described above with standard protein detection techniques, such as Western blotting or immunoprecipitation with an NRAGE-specific antibody (for example, the NRAGE antibody described herein). Compounds that modulate the level of NRAGE may be purified, or substantially purified, or may be one component of a mixture of compounds such as an extract or supernatant obtained from cells (Ausubel et al, supra). In an assay of a mixture of compounds, NRAGE expression is measured in cells ac-ministered progressively smaller subsets of the compound pool (e.g., produced by standard purification techniques such as HPLC or FPLC) until a single compound or nrinimal number of effective compounds is demonstrated to NRAGE expression.

Compounds may also be screened for their ability to modulate NRAGE biological activity (e.g., by preventing the interaction between NRAGE and p75NTR, E2F, necdin, the 'MAGE-like' protein, or another NRAGE protein). In this approach, the degree of NRAGE biological activity in the presence of a candidate compound is compared to the degree of biological activity in its absence, under equivalent conditions. Again, the screen may begin with a pool of candidate compounds, from which one or more useful modulator compounds are isolated in a step-wise fashion. Biological activity may be measured by any standard assay, for example, those described herein.

Another method for detecting compounds that modulate the activity of NRAGE is to screen for compounds that interact physically with NRAGE or that prevent the interaction between NRAGE and p75NTR, E2F, necdin, the 'MAGE- like' protein, or another NRAGE protein. These compounds may be detected, for example, by adapting interaction trap expression systems known in the art. These systems detect protein interactions using a transcriptional activation assay and are generally described by Gyuris et al. (Cell 75:791-803, 1993) and Field et al, (Nature 340:245-246, 1989), and are commercially available from Clontech (Palo Alto, CA). Alternatively, NRAGE or a fragment thereof can be detectably labeled and incubated with a candidate molecule. For protein-protein interaction studies, a second protein (e.g., necdin, p75NTR, 'MAGE-like') is also included. Following an appropriate incubation time, association of the compound with NRAGE (or a dissociation of NRAGE from the protein complex) is assayed. Compounds or molecules that function as modulators of NRAGE biological activity may include peptide and non-peptide molecules such as those present in cell extracts, mammalian serum, or growth medium in which mammalian cells have been cultured. A molecule that promotes a decrease in NRAGE expression or biological activity is considered particularly useful in the invention; such a molecule may be used, for example, as a therapeutic to decrease the ability of NRAGE to prevent cell proliferation. There is a tight association between cell proliferation and cell death. In some cases, a cell that receives a signal to proliferate will instead undergo apoptosis, presumably because the proliferation is inappropriate. Hence, depending on the context, a molecule that decreases NRAGE expression or biological activity may result in increased proliferation or, alternatively, cell death.

A molecule that increases NRAGE activity (e.g., by increasing dissociation from p75NTR) may be used to decrease cellular proliferation. This would be advantageous in the treatment of neoplasms or other cell proliferative diseases.

Antagonists or inhibitors of NRAGE may be administered at the site where nerve damage has occurred to stimulate nerve cell reconnection, division, and/or migration following accidental destruction of the nerve tract. Antagonists or inhibitors of NRAGE may reverse the differentiated state of neurons and allow new nerve connections to form.

It is likely that modulation of the interaction between any two MAGE proteins will modulate their function. Thus, compounds capable of disrupting the formation or stabilization of MAGE oligomers are useful for modulating any

MAGE biological activity (e.g., the NRAGE biological activity described herein). Compounds can be screened for their ability to modulate MAGE-MAGE interactions in a manner similar to that described for modulators of NRAGE- p75NTR interactions.

Therapy

To add NRAGE protein to cells in order to modulate cell proliferation, it is necessary to obtain sufficient amounts of pure NRAGE protein from cultured cell systems that can express the protein. Delivery of the protein to the affected tissue can then be accomplished using appropriate packaging or administrating systems. Alternatively, small molecule analogs may be used and administered to act as NRAGE agonists or antagonists and in this manner produce a desired physiological effect. Methods for finding such molecules are provided herein. Gene therapy is another potential therapeutic approach in which normal copies of the NRAGE gene or nucleic acid encoding NRAGE sense RNA are introduced into cells to successfully produce NRAGE protein, or NRAGE antisense RNA is introduced into cells that express excessive normal or mutant NRAGE. The gene must be delivered to those cells in a form in which it can be taken up and encode for sufficient protein to provide effective function.

Retroviral vectors, adenoviral vectors, adenovirus-associated viral vectors, or other viral vectors with the appropriate tropism for cells involved in a cell proliferation disease may be used as a gene transfer delivery system for a therapeutic NRAGE gene construct. Numerous vectors useful for this purpose are generally known (Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis and Anderson, BioTechniques 6:608-614, 1988; Tolstoshev and Anderson, Curr. Opin. Biotech. 1:55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et al, Nucl. Acid Res. and Mol. Biol. 36: 311-322, 1987; Anderson, Science 226: 401-409, 1984; Moen, Blood Cells 17: 407-416, 1991; Miller et al, Biotech. 7: 980-990, 1989; Le Gal La Salle et al, Science 259: 988-990, 1993; and Johnson, Chest 107: 77S-83S, 1995).

Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al, N. Engl. J. Med 323: 370, 1990; Anderson et al, U.S. Patent No. 5,399,346). Non- viral approaches may also be employed for the introduction of therapeutic DNA into cells otherwise predicted to undergo excessive or abnormal proliferation. For example, NRAGE may be introduced into a cell by lipofection (Feigner et al, Proc. Natl. Acad. Sci. USA 84: 7413, 1987; Ono et al, Neurosci. Lett. 117: 259, 1990; Brigham et al, Am. J. Med. Sci. 298:278, 1989; Staubinger et al, Meth. Enzymol. 101:512, 1983), asialorosonucoid-polylysine conjugation (Wu et al, J. Biol. Chem. 263: 14621, 1988; Wu et al, J. Biol. Chem. 264: 16985, 1989); or, less preferably, micro- injection under surgical conditions (Wolff et al, Science 247: 1465, 1990).

Gene transfer could also be achieved using non-viral means requiring infection in vitro. This would include calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for delivery of DNA into a cell. Although these methods are available, many of these are of lower efficiency.

Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods which are well known in the art. Transplantation of normal genes into the affected cells of a patient can also be useful therapy. In this procedure, a normal NRAGE gene is transferred into a cultivatable cell type, either exogenously or endogenously to the patient. These cells are then injected into the targeted tissue(s). In the constructs described, NRAGE cDNA expression can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in tumor cells may be used to direct

NRAGE expression. The enhancers used could include, without limitation, those that are characterized as tissue- or cell-specific in their expression. Alternatively, if an NRAGE genomic clone is used as a therapeutic construct (for example, following isolation by hybridization with the NRAGE cDNA described above), regulation may be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

Antisense based strategies may be employed to explore NRAGE gene function and as a basis for therapeutic drug design. The principle is based on the hypothesis that sequence-specific suppression of gene expression can be achieved by intracellular hybridization between mRNA and a complementary antisense species. The formation of a hybrid RNA duplex may then interfere with the processing/transport/translation and/or stability of the target NRAGE mRNA. Antisense strategies may use a variety of approaches including the use of antisense oligonucleotides and injection of antisense RNA. Antisense effects can be induced by control (sense) sequences, however, the extent of phenotypic changes are highly variable. Phenotypic effects induced by antisense effects are based on changes in criteria such as protein levels, protein activity measurement, and target mRNA levels. Such technology is now well known in the art, and sense or antisense oligomers or larger fragments, can be designed from various locations along the coding or control regions of sequences encoding NRAGE. In one example, the complementary oligonucleotide is designed from the most unique 5' sequence and used either to inhibit transcription by preventing promoter binding to the upstream nontranslated sequence or translation of an NRAGE-encoding transcript by preventing the ribosome from binding. Using an appropriate portion of the signal and 5' sequence of NRAGE, an effective antisense oligonucleotide includes any 15-20 nucleotides spanning the region which translates into the signal or 5' coding sequence of the polypeptide as shown in Fig. 1A. For example, NRAGE gene therapy may also be accomplished by direct administration of antisense NRAGE mRNA to a cell that is expected to undergo undesired apoptosis. The antisense NRAGE mRNA may be produced and isolated by any standard technique, but is most readily produced by in vitro transcription using an antisense NRAGE cDNA under the control of a high efficiency promoter (e.g., the T7 promoter). Adrnimstration of antisense NRAGE mRNA to cells can be carried out by any of the methods for direct nucleic acid administration described above.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples which may be used include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding NRAGE. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding NRAGE. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

An alternative strategy for inhibiting NRAGE function using gene therapy involves intracellular expression of an anti-NRAGE antibody or a portion of an anti-NRAGE antibody. For example, the gene (or gene fragment) encoding a monoclonal antibody that specifically binds to NRAGE and inhibits its biological activity may be placed under the transcriptional control of a cell type-specific gene regulatory sequence.

Another therapeutic approach within the invention involves administration of a recombinant NRAGE polypeptide (e.g, the ones described herein), either directly to the site of a potential or actual cell proliferation or apoptotic event (for example, by injection) or systemically (for example, by any conventional recombinant protein adrninistration technique). The dosage of NRAGE depends on a number of factors, including the size and health of the individual patient, but, generally, between 0.1 mg and 100 mg inclusive are administered per day to an adult in any pharmaceutically acceptable formulation. In other embodiments, any of the therapeutic proteins, antagonists, antibodies, agonists, antisense sequences or vectors described above may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of NRAGE, antibodies to NRAGE, mimetics, agonists, antagonists, or inhibitors of NRAGE. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be adininistered to a patient alone, or in combination with other agents, drugs or hormones. Methods well known in the art for making formulations are found, for example, in Remington: The Science and Practice of Pharmacy, supra. 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 NRAGE modulatory compounds 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 adrninistration in the form of nasal drops, or as a gel.

Diagnostics

Antibodies which specifically bind NRAGE may be used for the diagnosis of conditions or diseases characterized by expression of NRAGE, or in assays to monitor patients being treated with NRAGE, agonists, antagonists or inhibitors. The antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for NRAGE include methods which utilize the antibody and a label to detect NRAGE in human body fluids or extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules which are known in the art may be used, several of which are described herein.

A variety of protocols including ELISA, RIA, and FACS for measuring NRAGE are known in the art and provide a basis for diagnosing altered or abnormal levels of NRAGE expression. Normal or standard values for NRAGE expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to NRAGE under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, but preferably by photometric, means. Quantities of NRAGE expressed in subject, control and disease, samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.

The nucleic acid sequences encoding NRAGE may also be used for diagnostic purposes. The nucleic acid sequences which may be used include antisense RNA and DNA molecules, and oUgonucleotide sequences. The nucleic acid sequences may be used to detect and quantitate gene expression in biopsied tissues in which expression of NRAGE may be correlated with disease. The diagnostic assay may be used to distinguish between absence, presence, and excess expression of NRAGE, and to monitor regulation of NRAGE levels during therapeutic intervention. Nucleic acid sequences encoding NRAGE may be used for the diagnosis of conditions or diseases which are associated with expression of NRAGE. Examples of such conditions or diseases include Alzheimer's disease and cancers of the brain, prostate, breast, thyroid, skull, colon, gall bladder, kidney, lung, liver, small intestine, paraganglion, bladder, tongue, parathyroid, penis, and pancreas. The nucleic acid sequences encoding NRAGE may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pLN, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered NRAGE expression. Such qualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding NRAGE may be useful in assays that detect activation or induction of various cancers. The nucleotide sequences encoding NRAGE may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable control sample, the nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding NRAGE in the sample indicates the presence of the associated disease. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated with expression of NRAGE, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, which encodes NRAGE, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease. Once disease is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount of NRAGE transcript or polypeptide in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

The following examples are to illustrate the invention. They are not meant to limit the invention in any way.

Example 1: Isolation of NRAGE

We performed yeast two-hybrid screens to identify molecules that directly interact with the p75NTR intracellular domain. From this, an interacting protein, termed NRAGE, was identified. The characterization of the in vitro and in vivo activities of this molecule is described herein.

The initial cDNA identified in the two-hybrid screen was used to screen a rat MAH cell cDNA library, leacling to the identification of three overlapping cDNAs. The longest clone, 2.6 kilobases in length, detects a single mRNA species of 2.7 kb on Northern blots and contains a single open reading of 775 amino acids. In addition to the rat sequence, we have cloned human NRAGE cDNA. NRAGE is highly conserved between these two species, with over 90% of amino acid residues conserved. Similarity searches revealed that NRAGE is a member of a group of proteins termed the MAGE family (for melanoma associated antigen; see Fig. 2) whose functions remain largely unknown (see below). Like the MAGE family members, NRAGE contains a highly conserved 200 amino acid MAGE Homology Domain (MHD). Many genes of the MAGE family consist of tittle more than this conserved domain but some, including NRAGE, are much larger proteins (Fig. 2). Bootstrap homology analysis of NRAGE reveals that it is most closely related to a member of the MAGE family called Necdin. Structural features of NRAGE (which is an acronym for p_75 receptor interacting MAGE) are shown in Fig. 2. In addition to similarity to the MAGE family, the protein contains an extended stretch of proline-rich tandem repeats that show a low degree of similarity to helical heptad repeats present in neurofilaments.

Example 2: Expression pattern of NRAGE We have produced polyclonal antibodies against NRAGE using standard techniques and found that the molecule is expressed at high levels in a number of developing tissues but at much lower levels in the adult (Fig. 3). Northern blot analysis reveals that NRAGE mRNA is re-expressed in cell lines derived from a number of solid tumors.

Several members of the MAGE family show similar expression pattern to that of NRAGE, with high levels found early in development, low levels observed in most adult tissues, and increased expression levels within a variety of tumor types. Expression of MAGE family members within malignant cells results in their processing and presentation on the cell surface as peptide MHC antigens which are reactive to cytolytic T cells, and the first identified MAGE proteins were identified by virtue of their tumor-specific processing and expression as MHC -related peptides. The normal intracellular role of these proteins remains poorly studied and essentially unknown. A different developmental expression pattern is typified by Necdin.

Necdin is expressed at very low levels in neuronal precursors but its expression rises dramatically as these cells become post-mitotic. This expression pattern suggested that Necdin may play some role in the maintenance of the post-mitotic state. Consistent with this, forced Necdin expression in 3T3 fibroblasts arrested cell cycle progression. Deletion of the necdin gene is the cause of Prader-Willi syndrome, a relatively common neurogenetic disorder which results in hyperphagia and mental retardation, underscoring a role for Necdin in human nervous system development.

Necdin binds and functionally inactivates E2F-1 and can functionally compensate for the lack of pRB. The domain within E2F-1 that binds Necdin overlaps the domain responsible for binding pRB (Fig. 4). NRAGE and Necdin contain very similar MHDs and we have therefore asked if NRAGE also binds to the E2F transcription factors. Fig. 5 shows that, in vitro, NRAGE binds all members of the E2F family examined. It is likely that NRAGE also physically interacts with E2F factors in vivo and modulates their function. Example 3: Cellular localization of NRAGE

To assess the functional role of NRAGE, we have examined its cellular distribution, using epitope- and GFP -tagged forms of the protein. Necdin was predominantly localized to the nucleus. In contrast, tagged NRAGE was excluded from the nucleus (Fig. 6). These data are consistent with biochemical fractionation studies that showed that endogenous, untagged NRAGE was also excluded from the nucleus.

Example 4: NRAGE-mediated degradation of E2F through the ubiquitination- proteosomal pathway The controlled degradation of cell cycle proteins plays a key role in the regulation of cell cycle transit. E2F proteins are ubiquinated and subsequently degraded through the proteosomal pathway and a C-terminal domain (E2F-C) responsible for this rapid degradation has been identified in E2F-1. Deletion of this region greatly prolongs E2F-1 half-life and GAL4-E2F-C chimeras, into which this "degradation domain" has been introduced, show greatly accelerated proteolysis. A role for the regulated degradation of the E2F proteins have been suggested by recent studies that have found that binding of pRB to E2F-1 greatly prolongs E2F half-life. It is possible that C-terminal domains of E2F proteins contain a constitutive degradation signal which, when masked, results in rapid proteolysis or alternatively, the "degradation domain" may interact with a protein that hastens E2F-1 proteolysis. The identified "degradation domain" contains binding sites for pRB family members and for Necdin. Given its nuclear distribution, however, Necdin is unlikely to facilitate E2F degradation

We hypothesize that NRAGE may facilitate E2F degradation. NRAGE overexpression results in a reduction in the steady state levels of overexpressed

E2F-1, E2F-2 and E2F-3 (Fig. 7). Additionally, it is difficult to overexpress NRAGE at even moderate levels in most cell lines because it is rapidly degraded through the proteosomal pathway (Fig. 8). In contrast, deletion mutants of NRAGE lacking the C-terminal portion of the protein can be readily expressed at high levels and accumulates in the nucleus. NRAGE does not have a nuclear localization signal and the accumulation of the deletion mutant in this compartment is likely to be due to its association with a nuclear factor, possibly E2F, and suggests that the carboxy half of the molecule (a domain lacking in Necdin) precludes nuclear entry.

To identify the specific domain of NRAGE required for the interaction between E2F proteins and the carboxy terminal of NRAGE, GST fusion proteins containing successively smaller portions of the NRAGE interaction domains are produced by PCR and tested for their ability to interact with ³⁵S-E2F- 1 produced by in vitro translation (IVT). The minimal binding domain identified is then deleted from full length NRAGE (termed NRAGE"*) to produce a new construct (NRAGE^"E2F ). Each of these proteins is subsequently produced by IYT. Using standard techniques, pulldown assays are performed by incubating ³⁵S-E2F-1 with either NRAGE * and NRAGE^"E2F followed by immunoprecipitation with anti-NRAGE antibodies to confirm loss of binding between E2F-1 and NRAGE^{" E2F}. Pulldown assays are also used to test binding of NRAGE™¹ and NRAGE^'E2F to E2F- 2, -3 , - 4 and -5 in vitro.

To define the minimal domain within E2F-1 that is responsible for binding NRAGE, E2F-1 constructs with successive deletions in the carboxy terminus are tested as described above. HA-tagged E2F-1 with successive carboxy temύnal deletions is produced by IVT and then tested for interactions with the carboxy- terminal portion of the NRAGE molecule fused to GST using in vitro pulldown assays. Similar experiments are also performed using HA-tagged E2F-2 to test if NRAGE binds similar domains in both E2F proteins, a likely event since the carboxy termini of E2F-1 through -5 are very highly conserved.

To corifirm that the identified binding domains function to mediate NRAGE-E2F binding in vivo, myc-tagged NRAGE™¹ and NRAGE^"E2F are transiently expressed in SAOS-2 cells (which lack pRB) together with an HA- tagged E2F protein. In parallel experiments, myc-tagged NRAGE™¹ and HA- tagged E2F-1 mutants with successive carboxy terminal deletions are also transiently expressed in SAOS-2 cells. An anti-myc antibody is first used to immunoprecipitate either NRAGE™¹ or NRAGE^"E2F. E2F proteins are co- immunoprecipitated are then identified by immunoblotting using an anti-HA antibody (e.g., 12CA5, Babco Immunochemicals).

We produced antibodies to NRAGE by injecting rabbits with glutathione- S-transferase fused to NRAGE peptide fragments. Rabbits were injected with the same peptide two weeks later to improve the titer, and sera were collected at three, five, and seven weeks later. The antibodies specifically recognize human, rat, and mouse NRAGE on immunoblots and in irnmunocytochemistry.

NRAGE overexpression reduces cellular E2F levels, possibly by accelerating E2F degradation through the ubiquitin/proteosome pathway. PC 12 cells, which express detectable levels of NRAGE, were incubated with increasing concentrations of lactacystin, a specific proteosome inhibitor. Changes in the steady state levels of NRAGE were assessed by immunoblot using anti-NRAGE antibodies. As predicted, steady-state NRAGE levels were increased with lactacystin treatment (Fig. 8).

We have found that it is difficult to express exogenous NRAGE at high levels in most commonly-used cell types. Moreover, deletion analysis has revealed that an NRAGE mutant lacking amino acids 322 to 456 of NRAGE (NRAGE¹*⁰) accumulates to considerably higher levels than NRAGE™¹. We suspect that the domain deleted in NRAGE¹¹⁰ may be required for rapid proteosomal degradation that, in turn, may be required for E2F degradation. To confirm this, ³⁵S-Cys/³⁵S-Met pulse-chase analysis is first performed on a panel of NRAGE mutants in transfected COS 7 cells (which have very low levels of endogenous NRAGE). The effect of NRAGE on E2F protein turnover is then determined by exaπύning if overexpression of NRAGE™¹ or NRAGE^"E2F changes endogenous E2F turnover or E2F-1 ubiquination using methods described herein. We hypothesize that NRAGE™¹ will accelerate E2F degradation but that NRAGE^"E2F, which does not bind E2F proteins, will have no effect on E2F turnover. To test whether the rapid degradation domain of NRAGE is required for NRAGE-mediated E2F turnover, NRAGE^11® mutants, which lack this domain but contain the E2F binding domain, are examined for their ability to accelerate E2F turnover using methods described herein. It is likely that NRAGE^11® mutants will bind to E2F, but not accelerate E2F turnover. NRAGE inhibits passage through the cell cycle, presumably by reducing levels of E2F proteins. To examine this in more detail, the relative effect of overexpressing NRAGE™¹, NRAGE^11®, or NRAGE-^E2F on cell cycle transit, is measured by BrdU incorporation and NRAGE immunoflourescence, in 293 cells and SAOS-2 cells (which are pRB deficient). We hypothesize that NRAGE^11® and NRAGE^"E2F have no effect on cell cycle transit whereas NRAGE™¹ inhibits cell cycle transit in both cell lines.

To determine if NRAGE mediates its effect specifically through specific E2F proteins, the effects of NRAGE overexpression in fibroblasts lacking functional E2F-1 are determined. For this, fibroblasts from mice which lack both alleles of E2F-1 and from Svl29J control mice are prepared, transfected with NRAGE™¹, NRAGE^11®, or NRAGE-^E2F, and measured for the effects on cell cycle transit.

NRAGE and pRB likely compete for overlapping binding domains on E2F-1 and thereby regulate E2F protein levels. To assess whether NRAGE and pRB antagonize one another's E2F bmding activity, it is first determined whether pRB (produced by IVT) reduces the ability of a GST-NRAGE fusion protein to interact with ³ S-E2F-1 (also produced by IVT). A mutant form of pRB, lacking its E2F binding site, is used as a control in these experiments. Next, it is determined whether pRB and NRAGE antagonize or complement each others activity in vivo. For this, COS7 cells are transfected with plasmids encoding NRAGE together with increasing concentrations of pRB expression plasmid. The turnover rate of endogenous E2F is determined by ³⁵S-Cys/³⁵S-Met pulse- chase analysis, while cell cycle progression is determined by BrdU incorporation. The role of NRAGE on E2F activity can also be assessed in vivo, using

SAOS-2 cells, pRB-deficient osteocarcoma cells whose growth is inhibited by reintroduction ofpRB. pRC-CMV plasmids driving NRAGE™¹, NRAGE^11®, NRAGE^'E2F, or pRB expression are first stably transfected into SAOS-2 cells and colonies are selected using standard cell culture techniques. Selected colonies are then analyzed by immunoblot to confirm expression of each of the transfected proteins and to determine cellular levels of E2F-1. A similar experimental design can be used to assess the cellular outcome of cells that have been co-transfected with pRC-CMV plasmids driving NRAGE™¹, NRAGE^11®, NRAGE^"E2F and pCMV-hygroplasmids driving expression of pRB.

Example 5: NRAGE interacts with p75NTR

As described herein, NRAGE was identified by virtue of its interaction with the p75NTR intracellular domain. We first demonstrated this interaction in yeast, using the two-hybrid system. We have also demonstrated this interaction in vitro; in vitro translated NRAGE binds to GST-p75NTR, but not to GST alone (Fig. 9). Additionally, co-immunoprecipitations show that NRAGE and p75NTR interact when both are overexpressed in 293 cells (Fig. 10), and that endogenous NRAGE and p75NTR interact in PC 12 cells in the absence of neurotrophin.

In many signaling events, receptor activation causes aggregation of intracellular domains and subsequent localization of cytosolic signaling particles to the plasmalemma. For example, ligand-mediated aggregation of TNF receptor superfamily members results in binding of cytosolic TRAF and TRADD protein that, in turn, aggregate and activate downstream kinases. Alternatively, some receptors associate constitutively with cytosolic molecules which are released following receptor activation. In one example, the "silencer of death domain" (SODD) protein is constitutively bound to the TNF-R1 and DR3 receptors and is released upon receptor activation. Our data are consistent with the hypothesis that p75NTR binds NRAGE constitutively and that NRAGE is released from the receptor following activation. It is likely that NRAGE is released from p75NTR as a function of neurotrophin binding, resulting in a rise of cystolic NRAGE levels. NRAGE translocation to the cytosol may then compete with pRB family members for common E2F binding sites. We further hypothesize that cytosolic NRAGE will reduce cellular levels of E2F proteins. p75NTR activation would therefore be expected to favor cell cycle exit or stabilization of a non- proliferative state. The proliferative phase of neuronal loss occurs early in development. In this phase, neuroblasts leave the cell cycle and either become viable post-mitotic neurons or die. Neuronal cell death is widespread during this phase. The connection between p75NTR, NRAGE and E2F proteins, described herein, raises the possibility that a p75NTR-NRAGE pathway regulates cell cycle/apoptotic events during the proliferative phase of neuronal loss. To test this, we have examined the effect of NRAGE overexpression on cell cycle control in COS7 and 293 A cells by BrdU labeling and found that NRAGE has a profound inhibitory effect on cell cycle transit in these two lines. Other work directly supports a role for p75NTR in apoptotic events that are linked to the cell cycle. Apoptosis within the developing spinal cord at E10.5, at the peak phase of neurogenesis, occurs both within the ependymal zone, which consists mostly of neuronal precursors, and within the mantle zone, which contains newly born neurons. In p75NTR -/- mice, the incidence of apoptosis in the spinal cord at this time is sharply reduced, particularly in the mantle zone. It is not known if these cells maintained in the spinal cord of p75NTR -/- remain viable, but increased numbers of basal forebrain cholinergic and sympathetic neurons have been noted in the p75NTR -/- mouse, and it is possible that these supernumerary neurons arise due to a defect in cell cycle exit or apoptosis. Other data consistent with a role for p75NTR in the phase of neuronal development or loss has emerged from detailed analysis of NT3-/- mice. The majority of wild-type DRG neuroblasts exit the cell cycle at E11/E12 to form postmitotic neurons, while NT-3-/- neuroblasts precede through the normal Gl restriction point and die by apoptosis in S phase. These dying neurons show dramatic elevations in E2F-1 levels, as well as in levels of several Gl -specific proteins that are regulated by E2F transcriptional activity. This early phase of cell death is not observed in trkC-/- mice. As a result, the NT3-/- mice have about 50% fewer DRG neurons at P0 than do trkC-/- mice. These data suggest that NT-3 normally acts to suppress mitogenesis and/or apoptosis in the developing DRG by acting through a receptor other than trkC. p75NTR is expressed in essentially all of the sensory precursors at this stage of DRG development and it is likely that p75NTR, which is abundantly expressed in the DRG neurons at this stage, attenuates cell cycle progression and reduces apoptosis in the developing DRG. Another possibility is that NT-3 acts through trkA to mediate these effects. This is unlikely since NT-3 does not support survival through trkA in the developing DRG (White, 1996) and since NT-3 is a poor ligand for trkA, particularly when trkA is co-expressed with p75NTR. In chick embryos treated with exogenous NGF, the proportion of sympathetic neuron precursors in S-phase is nearly double that of controls, strongly suggesting that NGF acts either as a mitogenic factor (Goldstein et al

1997) or slows cell cycle exit. Since trkA is not present in the avian sympathetic ganglia until later in development, these early effects of exogenous NGF are most likely mediated by p75NTR. Together, these recent studies raise the possibility that p75NTR may help regulate cell cycle and apoptotic events as neuroblasts make the transition to a post-mitotic state.

Example 6: The effect of neurotrophin binding p75NTR on NRAGE translo cation and NRAGE-E2F interactions

The analysis of the interaction of p75NTR with downstream signaling molecules and its regulation is complicated by the fact that there are at least four neurottophins capable of binding p75NTR and exerting differential effects.

Furthermore, p75NTR is often co-expressed with members of the trk family and there is extensive crosstalk between p75NTR and trk receptors, with p75NTR regulating trk activity in some circumstances and trk regulating p75NTR in others. p75NTR regulates the cellular location of NRAGE. In cells lacking p75NTR, NRAGE is predominantly located in the cytosol. When co-expressed with p75NTR, however, NRAGE is located mainly at the plasma membrane. Addition of neurotrophin to cultures of p75NTR-expressing cells causes a reduction in the plasma membrane-associated NRAGE, and an increase in cytosohc NRAGE. When p75NTR and trkA are co-expressed, they form a stable complex. NRAGE fragments disrupt this complex. This suggests that when p75NTR and trkA are coexpressed, they form a complex that requires NRAGE. To unravel these complex interactions, a comprehensive analysis of the effect of the various neurotrophins on the association of NRAGE with p75NTR in PC 12nnr cells, which express both p75NTR and NRAGE but lack trk expression, can be performed. Immunoprecipitates of p75NTR before and after stimulation with one of the four neurotrophins for period up to two hours and at concentrations up to 250 ng/ml are analyzed for the presence of NRAGE. In parallel experiments, immunoprecipitates of NRAGE following the same treatment conditions are analyzed for the presence of p75NTR. It is very likely that the NRAGE-p75NTR complex is differentially affected by the various neurofrophins. To determine how the trk receptor contributes to NRAGE mobilization, similar experiments are performed using this cell system before and after infection with recombinant adenovirus encoding either wild-type trkA, kinase-dead trkA or lacZ (used as a negative control), followed by analysis of NRAGE translocation. Preferably, untreated and neurotrophin-treated PC12nnr5 cells are processed by differential centrifugation to separate membrane, nuclear, and cytosolic compartments. Each or these are analyzed for NRAGE content by immunoblot. Finally, an immunocytochemical approach is used to evaluate localization of NRAGE in intact cells. Myc- and GFP -tagged forms of NRAGE show identical distributions when expressed in COS7 and 293A cells, indicating that tagging does not affect NRAGE distribution. We can examine the distribution of tagged NRAGE™¹ in the presence and absence of p75NTR and in the presence and absence of neurotrophin, using standard immunocytochemical techniques. We can also examine the subcellular distribution of untagged NRAGE using affinity purified anti-NRAGE antibodies.

To confirm that p75NTR activation regulates the cell cycle through an NRAGE pathway, SAOS-2 cells are transfected with p75NTR and either NRAGE™* or control plasmids; cells are then treated with neurotrophin and the percentage of transfected cells passing through S phase is quantified by BrdU incorporation. PC12nnr5 cells endogenously express both p75NTR and NRAGE. Using FACS analysis, one can analyze proportions of cells in Gl, G2, M, and S phase and thus determine whether neurofropliin-mediated p75NTR activation can affect cell cycle kinetics in these cells. It is likely that NRAGE^11® or NRAGE^'E2F interferes with the action of endogenous NRAGE by acting in a dominant inhibitory fashion.

Overexpression of p75NTR in a 3T3 derivative line results in a dramatic reduction in colony formation (Figs. 11A-11C). It is likely that this is due to an effect on growth suppression, rather than a direct activation of an apoptotic pathway.

To determine if p75NTR plays some role in cell cycle progression in mauiring neuroblasts, we can examine the developing spinal cord and DRG of p75NTR -/- animals for defects in appropriate cell cycle exit, expression of cell cycle marker genes and level and distribution of NRAGE protein. p75NTR -/- mice were produced on a background consisting of a mixture of Svl29 and Balb/c strains. To allow comparisons in appropriate genetic backgrounds, p75-/- mice are back-crossed to wild-type mice from the Svl29 strain. Heterozygous FI progeny are then inbred to produce F2 progeny that are p75NTR+/+, p75NTR+/- and p75NTR-/- and these F2 progeny are analyzed using any of the techniques described herein. Using standard techniques, we can produce mice in which the NRAGE allele has been rendered null. A homologous recombination vector is produced, targeting, for example, the exon containing the initiator methionine of NRAGE. This targeting vector is then electroporated into ES cells and clones appropriate for blastocyst injection are analyzed. Heterozygous FI progeny are bred to produce NRAGE+/+, NRAGE+/- and NRAGE-/- animals.

Overexpression of the intracellular domain of p75NTR results in losses of central and peripheral neurons during development. The promoter driving expression of the p75NTR intracellular domain in these animals is first active precisely at the time neurons are exiting the cell cycle. Neuronal loss observed in these animals is likely due to inappropriate regulation of NRAGE and the E2F pathway, since we have found that lysates derived from El 8 cortex of p75NTR intracellular domain overexpressors contain less NRAGE and more E2F-1 protein compared to their wild-type littermates.

Example 7: Relationship of other MAGE family members to NRAGE biological activity

The methods for identifying compounds that modulate NRAGE expression or biological activity can also be used to identify compounds that modulate the expression or activity of other MAGE family members. The discovery of NRAGE and its interaction with both p75NTR and E2F family members provides a link between the cell surface and cell cycle control, but these discoveries can be extended to other members of the MAGE family. There is a high degree of sequence similarity shared between NRAGE and other MAGE family members. Since one highly-conserved region is the domain responsible for interactions with E2F proteins, it is very likely that the E2F interactions we describe for NRAGE will also be true for other MAGE family members. Most, if not all, MAGE family members are expressed at high levels in a wide variety of tumor cells. It is reasonable to expect that MAGE family members may contribute to growth control within cancerous cells and, thus, may be useful targets for therapeutics with which to modulate cell proliferation. NRAGE and Necdin each acts as a tumor suppressor, and it is likely that additional MAGE family members also inhibit or prevent cell cycle progression. It is also likely, however, that some MAGE family members act as antagonists of NRAGE, by stabilizing E2F proteins or enhancing E2F transcriptional activity, thereby promoting cell proliferation.

Example 8: NRAGE forms complexes with itself and other MAGE proteins in vitro We hypothesized that MAGE family members such as NRAGE, necdin, and 'MAGE-like' may complex with themselves. To test this in vitro, GST-fusion proteins containing the MAGE domain of individual MAGE family members are produced. ³⁵S-labeled MAGE proteins produced by in vitro translation are then be incubated with these GST-fusion proteins precoupled to glutathione sepaharose. After incubation, the GST-sepharose complexes are extensively washed (to remove non-specific interacting proteins), and retained proteins are eluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE and fluorography. Using the foregoing method, an interaction between GST-NRAGE and

'MAGE-like' was identified. Similarly, a heterophilic interaction has also been detected between GST-NRAGE and necdin. We have also detected a homophihc interaction between GST-mNRAGE and ³⁵S-NRAGE. Together, these results indicate that certain MAGE family members interact and form MAGE oligomers.

Other Embodiments

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

Claims

What is claimed is:

1. A method for identifying a compound that modulates cell proliferation, said method comprising:

(a) providing a cell expressing an NRAGE polypeptide and an E2F polypeptide;

(b) contacting said cell with a candidate compound; and

(c) monitoring the level of binding of said NRAGE polypeptide to said E2F polypeptide in said cell, wherein a change in said level of binding in the presence of said candidate compound, relative to a level of binding in a cell not contacted with said candidate compound, identifies said candidate compound as a compound that modulates cell proliferation.

2. The method of claim 1, wherein said cell is from a mammal.

3. The method of claim 2, wherein said mammal is a human or a rodent.

4. The method of claim 1, wherein said NRAGE polypeptide is human NRAGE.

5. A method for identifying a compound that modulates cell proliferation, said method comprising:

(a) providing an NRAGE polypeptide, an E2F polypeptide, and a candidate compound;

(b) contacting said NRAGE polypeptide, said E2F polypeptide, and said candidate compound; and

(c) monitoring the level of binding of said NRAGE polypeptide to said E2F polypeptide, wherein a change in said level of binding in the presence of said candidate compound, relative to a level of binding of said polypeptides not contacted with said candidate compound, identifies said candidate compound as a compound that modulates cell proliferation.

6. The method of claim 5, wherein said polypeptides are in a cell-free system.

7. The method of claim 6, wherein one of said polypeptides is immobilized.

8. The method of claim 5, wherein said NRAGE polypeptide and said candidate compound are first contacted, and then said E2F polypeptide is contacted with said NRAGE polypeptide and said candidate compound.

9. The method of claim 5, wherein said E2F polypeptide and said candidate compound are first contacted, and then said NRAGE polypeptide is contacted with said E2F polypeptide and said candidate compound.

10. The method of claim 5, wherein said NRAGE polypeptide and said E2F polypeptide are first contacted, and then said candidate compound is contacted with said NRAGE polypeptide and said E2F polypeptide.

11. A method for diagnosing a mammal for the presence of a cell proliferation disease or an increased likelihood of developing said disease, said method comprising measuring the level of binding of an NRAGE polypeptide to an E2F protein in a sample from said mammal, wherein an alteration in said level of said binding relative to a level of binding in a sample from an unaffected mammal being an indication that said mammal has said disease or increased likelihood of developing said disease.

12. The method of claim 11, wherein said mammal is a human or a rodent.

13. A method for modulating proliferation of a cell, said method comprising admiiustering to said cell a compound that modulates the binding of an NRAGE polypeptide to an E2F polypeptide.

14. The method of claim 12, wherein said compound is selected from the group consisting of a chemical, a drug, and an antibody that specifically binds to an NRAGE polypeptide.

15. The method of claim 14, wherein said antibody is a neutralizing antibody.

16. The method of claim 12, wherein said cell is in a mammal.

17. The method of claim 16, wherein said mammal is a human or a rodent.

18. The method of claim 16, wherein said modulating of proliferation of said cell is decreasing said proliferation and said mammal is diagnosed as having a cell proliferation disease.

19. The method of claim 12, wherein said compound is an NRAGE antisense nucleic acid molecule.

20. The method of claim 12, wherein said NRAGE polypeptide is a human NRAGE polypeptide.