WO1998006753A2

WO1998006753A2 - P53 mutant

Info

Publication number: WO1998006753A2
Application number: PCT/IB1997/001275
Authority: WO
Inventors: Arnold Jay Levine; Kristen Kay Walker
Original assignee: Trustees Of Princeton University
Priority date: 1996-08-13
Filing date: 1997-08-05
Publication date: 1998-02-19
Also published as: WO1998006753A3; AU4394697A

Abstract

The present invention relates to p53 mutants which have lost the ability interact with other proteins or compounds or to bind a SH-3 domain, as well as nucleic acids encoding these mutants, suitable expression systems and uses of such subjects matters, e.g. for the development of pharmaceutical agents. The invention further relates to peptides derivable from the proline rich domain of p53.

Description

P53 Mutant

The present invention relates to p53 mutants which have lost the ability to interact with other chemical entities in a cell that are important for p53 function, as well as nucleic acids encoding these mutants, suitable expression systems and uses of such subjects matters, e.g. for the development of pharmaceutical agents. The invention further relates to peptides derivable from the proline rich domain of p53.

The p53 tumor suppressor protein plays a pivotal role in the prevention of cellular transformation and tumorigenesis by curtailing the proliferation of cells harboring potentially oncogenic lesions. The unusually high frequency of p53 mutations observed in human cancers indicates the importance of the anti-proliferative pathways under p53 regulation. Indeed, evidence now suggests that p53 can respond to multiple signals of cellular alarm, including DNA damage, hypoxia, and perturbations of cell cycle regulation, by inducing either a transient growth arrest around the G1/S transition of the cell cycle or programmed cell death (apoptosis). The p53 gene resides on chromosome 17p.

At the molecular level, p53 has been demonstrated to function as a transcriptional activator. Transcriptional activation is dependent upon three independent structural domains which mediate (1 ) sequence-secific DNA binding (Y. Cho, S. Gorina, P. D. Jeffrey, N. P. Pavletich, Science 265, 346-355 (1994)), (2) interactions with the basal transcription factor TrllD, (C.J.Thut, J. L Chen, R. Klemin, R. Tjian, Science 267, 100-104 (1995); H. Lu, A. J. Levine, Proc. Natl. Acad. Sci. USA 92, 5154 (1995)) and (3) homooligomerization (G. M. Clore, et al., Science 265, 386 (1994)). That the induction of novel gene expression underlies p53'ε ability to alter cell cycle progression is strongly supported by evidence that several p53 target genes, such as p21 , MDM2, GADD45, and cyclin G, encode for potential cell cycle regulatory proteins. Moreover, in cell culture systems, p53 mutants engineered for the loss of transcriptional activation are also defective in activating the G1 cell cycle checkpoint (J. Martinez, et al., Genes & Development s, 151-159 (1991); P. W. Hinds, et al., Cell Growth & Diff: 1, 571-580 (1990)).

In contrast, the molecular mechanism underlying p53-dependent apoptosis is less well-defined. Recently, it has been demonstrated that p53-dependent apoptosis can proceed despite the inhibition of ongoing protein synthesis (C. Caelles, A. Heimberg, M. Karin, Nature 370, 220-223 (1994)) as well as in response to ectopic expression of p53 mutant protein that is transcriptionally inactive (A. J. Wagner, J. M. Kokontis, N. Hay, Genes & Devel. 8, 2817-2830 (1994); Y. Haupt, W. S. Alexander, G. Barri, S. P. Klinken, J. M. Adams, Ce//65, 753-763 (1991)).

The present invention is based on the identification of a new unique p53 structural and functional domain. Since said domain is characterized by a relative abundance of the amino acid proline, it is referred to as proline rich domain hereinbelow. In wild-type human p53 (393 amino acids), the proline rich domain extends approximately from aa residue 61 (Asp) to amino acid residue 94 (Ser). In said domain, 12 of 34 amino acid (aa) residues are proline residues, and it contains five repeats of the amino acid sequence P-X-X-P, P designating a proline residue and X designating any naturally occurring L-amino acid. This is the consensus binding motif for SH3 domains (R. Ren, B. J. Mayer, P. Cicchetti, D Baltimore, Science 259, 1157-1161 (1993); H. Yu, et al., Ce//76, 933-945 (1994); A. Mussachio et al., Nature Struct. Biol. 1, 546-551 (1994)). The P-X-X-P motif forms a left- handed helix when it binds to the SH3 domain binding site, thus enabling protein-protein interactions. The P-X-X-P motif has been conserved over evolution in p53 molecules of divergent species. For example, the monkey has three repeats of P-X-X-P within aa residues 61-94, mouse has two within aa residues 55-88, rat has one repeat within aa residues 60-92 and chicken has four repeats within aa residues 56-89 as determined by Soussi et al. {Oncogene 5, 945-952 (1990)).

Since many proteins that are encoded by oncogenes, contain SH3 domains, the presence of P-X-X-P repeats in p53 leads to the hypothesis that p53 can interact with other proteins via its proline rich domain and this in turn may have a functional consequence. As a step towards deeper insight into the mechanisms by which p53 can signal for apoptosis or regulation of cell division function we have investigated the role of the proline rich domain and the P-X-X-P motif in p53. There is a need for elucidating p53's role, e.g. for the identification of physiological and non-natural ligands that are capable of interacting with p53 via the proline rich domain and the P-X-X-P motif. Such knowledge is important for design and identification of more efficient and less toxic drugs beneficial in the treatment of possible p53- related diseases, such as neurodegenerative (via apoptosis) or proliferative diseases. It is an object of the present invention to fulfill this and other needs. Summarv of the invention

It is an object of the present invention to provide a p53 mutant protein which has a mutation in the proline rich domain said mutation affecting the specific interaction of p53 with another molecule or molecules via said domain.

In another embodiment the present invention relates to an (oligo-) peptide comprising the proline rich domain of p53 or at least two P-X-X-P repeats. In particular, the invention concerns such peptide, or a fusion protein comprising such peptide, whose overexpression in cells blocks p53 mediated activities - proliferative or degenerative.

The invention moreover provides a nucleic acid encoding such p53 mutant protein, or peptide, and fragments of such nucleic acids including oligonucleotides.

It is yet another object of the present invention to provide systems including nucleic acid expression vectors suitable for expression of the nucleic acids of the invention. For example, the overexpression of the proline rich domain by itself, in a cell, may have therapeutic value or block the action of p53, or related molecules.

It is still another object of the invention to provide a method for distinguishing between p53-mediated apoptosis and cell cycle arrest. The mutant protein acts as a transcription factor, but is at least partially or substantially defective in cell killing.

In a further aspect, the invention relates to assays suitable for identification of an agent capable of specific interaction with the proline rich region of p53. Thus, the present invention provides a method for discovering compounds able to activate the cellular pathway that induces molecules that interact with the proline rich domain of p53. Such a method rests upon activating the p53 wildtype, but not a proline domain mutant of the invention. Such a method involves e.g. exposing a compound or mixture of compounds to wildtype p53 and determining the ability of the compound or mixture to activate the p53 wild-type, and exposing said compound or mixture to a p53 mutant protein of the invention and determining the inability of the compound or mixture to activate said mutant. Alternatively, such method may involve use of a p53 peptide according to the invention plus or minus the proline rich domain.

Furthermore, the present invention provides a method for discovering or modulating a p53-mediated direct signaling pathway, and this method does not involve transcription by p53.

In yet another aspect, the present invention relates to the introduction into a cell of a p53 mutant nucleic acid, e.g. a p53 mutant cDNA, encoding a p53 mutant protein or (oligo-) peptide and then examining the resultant physiological impact, so as to develop agents that block apoptosis, initiate apoptosis or result in cell death.

Detailed description of the invention

In one embodiment, the present invention relates to p53 protein mutants or muteins differing from wild-type p53 protein by a mutation, particularly a deletion, in the proline rich domain of said protein, which mutation affects the capability of the protein to interact physically or otherwise with another molecule via said domain. In particular, the p53 mutation affects a p53-dependent signaling pathway and essentially abolishes, specific and selective interaction of the mutein to a protein domain which can be bound by wild-type p53 via its proline rich domain. The p53 mutant, according to the invention, is a recombinant protemexpressed in cells or obtainable through microbial expression, optionally in combination with chemical synthesis.

As used herein, "proline rich domain" means the contiguous stretch of ammo acids (and triplets encoding these) extending in wild-type p53 approximately between ammo acid residue 55 and ammo acid residue 94 and characterized by one, two, three, four or five copies of the P-X-X-P motif, wherein P denotes a proline residue and X may be any naturally occurring L-amino acid. As mentioned above, in human wild-type p53 the proline rich domain extends approximately from ammo acid 61 to am o acid 94 and contains five copies of the P-X-X-P motif. More extensive mutant analysis of the the p53 protein has shown that not all P-X-X-P motifs are equal in their physiological impact on a cell The last two P-X-X-P repeats (aa 82-94) are the most critical ones in p53 function.

For the purposes of the present specification, the term "wild-type p53" in particular means the nucleotide or ammo acid sequences reported by Matlashewski et al., EMBO J. 13, 3257-3262 (1984); Zakut-Han et al., EMBO J. 4, 1251 -1255 (1985); Lamb and Crawford, Mol. Cell. Biol. 5, 1379-1385 (1986); D. Pennica et al., Virology 134, 477-482 (1984), and A.J. Lev e, Ann. Rev. Biochem. 62, 623-651 (1993). Sequences are available from GenBank. Wild-type p53 includes a proline/arginine polymorphism at amino acid 72 and the corresponding nucleotide polymorphism.

The term "signaling pathway" refers to p53 biological acitivities which can be mediated via another protein and regulating one or more p53 dependent phenotypes without requiring p53-mediated transcription activity. The p53 exerts its signaling function via the above- defined proline rich domain. A mutant p53 protein according to the invention differs from wild-type p53 because it is unable to interact with a cellular protein resulting resulting in the fialure of a p53 dependent phenotype or function. In particular, in contrast to wild-type p53, a mutant of the invention does not to a significant extent interact with molecules required to mediated p53 apoptosis or G1 arrest. The SH3 domain which can bind to a P-X-X-P motif is one type of possible p53 interacting molecule and it is present in proteins involved in growth regulation. The SH3 domain in the c-abl protein binds to a p53 peptide which has the P-X-X-P repeats. The SH3 domain of the src protein does not bind to such p53 peptide, demonstrating specificity. The mutants of the invention have never been seen to occur naturally.

A p53 amino acid mutant as provided in the present invention may be a substitutional, insertional or deletional variant of wild-type p53. Substitutions, deletions and insertions in the proline rich domain of wild-type p53 may be combined to arrive at an amino acid mutant of the invention. The deletion mutations have been shown to affect one, some or all of the P-X-X-P motifs present in the proline rich domain. Preferred are deletional variants wherein optionally one or more amino acids are replaced with another amino acid. Particularly preferred are deletional mutants which lack essentially the complete proline rich domain, or deletional mutants lacking the last two P-X-X-P repeats.

Most preferred mutants of the invention are derivable from human wild-type p53 and include deletional mutants lacking at least two or more, e.g. three, four or five P -X-X-P motifs.

Examples of such preferred mutants are dl(64-67, 77- 80), dl(72-75, 77-80), dl(64-67, 89-92), and dl(72-75, 89-92). The nomenclature provides the deleted (dl) amino acid residues of mature human p53 in parenthesis with the amino-terminal residue=1. Particularly preferred is the p53 mutant designated dl(62-91) lacking aa residues 62-91 of human mature p53, i.e. lacking the proline rich domain including ail five P-X-X-P motifs at aa residues 64-67, 72-75, 77-80, 82-85, 89-92. Equally preferred is a p53 mutant lacking aa residues 82-85, 89-92, e.g. the mutant designated dl(82-94). In mutants of the invention having at least one P -X-X-P motif, one or more of the proline residues in the remaining proline rich domain may be replaced with another amino acid, preferably conservatively with a naturally occurring L-amino acid, such as alanine. For example, proline to alanine substitutions may be made at aa positions 77, 80, 82, 85, 89 and/or 92.

The (in-)ability of a p53 mutant to bind to a SH3 domain, e.g. an abl-SH-3 domain, is determined according to methods known in the art, e.g. in a conventional in vitro binding assay, employing e.g. a suitable GST fusion protein or a peptide linked to a column. Advantageously, such determination involves comparison to suitable controls, particularly comparison to wild-type p53, or a mutant thereof capable of selective binding to SH-3 containing proteins. For instance, mutants suitable as positive controls are the double point mutant L22Q-W23S and the tumor derived mutant R175H having a mutation at codon 175. This mutant binds better to c-abl than wild-type p53 (see Examples). Not every SH3 domain binding protein binds to p53. For example, the src or Crk oncogenes do not bind to p53. Such proteins are therefore not suitable to determine ability or inability of a p53 mutant to bind to a SH3 domain. The SH3 binding domain may be presented in an isolated form, as a fusion protein, e.g. as a fusion protein comprising glutathione S-transferase (GST), or as a naturally cccuπng protein Potential binding partners of p53 include the c-abl protein

The invention further comprises derivatives of a mutant of the invention Such derivatives are encompassed by the term " mutant of the invention" Derivatives of the invention include molecules wherein a mutant of the invention is covalently modified by substitution, chemical, enzymatic or other appropriate means with a moiety other than a naturally occurring ammo acid Such a moiety may be a detectable moiety such as an enzyme, a radioisotope or a fluorescent For example, derivatives include a covalent or aggregative conjugate of a mutant of the invention with another chemical moiety, said derivative displaying essentially the same biological activities as the undenvatized mutant of the invention

Derivatives of a mutant of the invention also comprise proteins which, as compared to wild-type p53, contain am o acid deletions, additions or substitutions outside the proline rich domain as defined above. Such modifications are subject to the requirement that the derivatives maintain the characteristic feature of the mutants of the invention. The modifications may be deliberate, as by site-directed mutagenesis, or spontaneous. Am o acid substitutions are typically of single residues, insertions usually will be on the order of from one to about ten ammo acid residues, and deletions will usually range from about one to about thirty residues Thus, conservative am o acid substitutions may be made substantially without altering the nature of the mutant of the invention. Modifications involving deletion of two or more consecutive am o acids in the p53 sequence result in fragments of the mutants of the invention which are also encompassed.

The mutants of the invention are useful in the identification of a compound which selectively binds to the proline rich region of p53, e.g. as a positive or negative control in an assay designed for this purpose. For example, the mutants of the invention are useful in the identification of physiological p53 hgands, e.g. proteins or other moieties specifically interacting with p53 through the P-X-X-P motif or the proline rich domain. Furthermore, a mutant of the invention is useful in a method for identifying a signal or agent which selectively activates p53, or which is capable of modulating the p53-mediated signaling pathway without involving transcription. Additionally, a mutant of the invention is useful for the generation antibodies. A mutant of the invention capable of competing with endogenous wild-type or mutant p53 protein for an endogenous ligand is envisaged as therapeutic agent, e.g. in the treatment of a neurodegenerative or proliferative disease.

Advantageously, a mutant of the invention is obtained by chemical synthesis and/or recombinant DNA techniques. Based on p53 amino acid sequence information chemical synthesis of a mutant of the invention may be performed according to conventional methods known in the art. In general, those methods comprise the sequential addition of one or more amino acid residues to a growing (poly)peptide chain. If required, potentially reactive groups, e.g. free amino or carboxy groups, are protected by a suitable, selectively removable protecting group. Chemical synthesis may be particularly advantageous for fragments of a mutant of the invention having no more than about 100 to 150 amino acid residues.

Alternatively, a mutant of the invention may be produced from a DNA encoding wildtype p53 which has been subjected to in vitro mutagenesis resulting in the desired deletion of amino acid encoding triplets in the proline rich domain (e.g. encoded approximately by bp 183 to 285 in human wild-type p53 cDNA) and, optionally, an addition or exchange of triplets. Also, based on the p53 sequence data commonly available, a person of ordinary skill in the art is able to apply standard hybridization technology to produce a mutant of the invention. For example, a mutant of the invention is obtainable using PCR to amplify cDNAs encoding selected p53 fragments. More specifically, a PCR-based method may be used to create an internal deletion of amino acids 62-91 , removing all five P-X-X-P motifs from the human p53 protein.

The invention also provides a method for preparing a mutant of the invention, said method being characterized in that suitable host cells producing the mutant of the invention are multiplied in vivo. Preferably, the host cells are transfected with a hybrid vector comprising an expression cassette comprising a promoter and a DNA sequence coding for a mutant of the invention which DNA is controlled by said promoter. Subsequently, the mutant of the invention may be recovered. Recovery comprises e.g. isolating the mutant of the invention in the host cells or isolating the host cells expressing the mutant. More specifically, the invention provides a method for producing a mutant of the invention which method comprises growing host cells transfected with a DNA construct comprising a DNA coding for said mutant of the invention, and optionally recovering the mutant. If desired, the host cells lack endogenous p53.

Generally, host cells suitable for production of a mutant of the invention include eukaryotic cells, e.g. animal cells, particularly mammalian cells. The vector containing the mutant gene can be propagated in prokaryotic cells, such as gram-positive and gram- negative bacteria, e.g. E. coli. A mutant of the invention can be produced directly in recombinant cell culture or as a fusion with a signal sequence, preferably a host- homologous signal. Higher eukaryotic host cells are preferred.

As used herein, in vitro means ex vivo. In vivo includes cell culture and tissue culture conditions, as well as living organisms, e.g. transgenic animals.

A mutant of the invention may be derivatized in vitro or in vivo according to conventional methods known in the art.

In another aspect, the present invention relates to peptides, which are capable of selectively binding to an SH-3 binding domain , e.g. the SH-3 binding domain of c-abl. Such peptides comprise a P-X-X-P motif, preferred peptides comprising from two to five P-X-X-P motifs. Particularly preferred peptides are such peptides comprising part of or essentially the complete proline rich domain of wild-type p53, e.g. the contiguous stretch of amino acid residues 61 to 94 of human wild-type p53, and typically containing from about 12 up to about forty, particularly from about 13 to about 34 consecutive amino acids of the contiguous stretch of amino acid residues 61 to 94 of human wild-type p53. Exemplary peptides include the peptide with the amino acid sequence set forth in SEQ ID NO:3/SEQ ID NO:4, and fragments of said peptide lacking one or more N-terminal and/or C-terminal amino acids. Fragments within said definition are e.g. the 13mer and 12mer peptides having the sequence extending from the amino acid at positions 22 (or 23) to the amino acid at position 34 in SEQ ID NO:3/SEQ ID NO:4. Advantageously, the peptides of the present invention are prepared by chemical synthesis according to conventional methods known in the art. The limitation on peptide size is primarily due to the size and purity limitations imposed by current technologies. Peptides of the invention also encompass derivatives as defined above, including peptides immobilized to a solid support, e.g. beads. The peptides of the invention may be used e.g. to induce, enhance or retard p53-dependent apoptotic pathways in a cell. The p53 proline rich domain mediates an activity which is crucial for the effective transmission of p53 anti-proliferative signals. Therefore, peptides of the invention, or suitable peptidomimetics, suitable for in vivo administration and capable of competing with endogenous p53 ligand to the proline rich domain are envisaged as therapeutic agents. Furthermore, such peptides may be useful for the identification of a compound that modulates the p53 mediated direct signal pathway. A peptidomimettc or a peptide analogue is e.g. a compound modelled to resemble the three-dimensional structure of a peptide of the invention as defined above. The designing of mimetics to a (pharmaceutically active) peptide is a known approach to the design of drugs based on a "lead" compound. This may be desirable e.g. where the "original" (active) peptide is difficult or expensive to synthesize, or where it is unsuitable for a particular mode of administration, e.g. peptides are considered unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary channel.

The invention further provides oligonucleotides encoding a peptide of the invention. Preferred are such oligonucleotides encoding peptide above designated as being preferred.

The invention also relates to the use of a mutant or peptide of the invention as antigen, i.e. for the generation of polyclonal and, preferably, monoclonal antibodies which specifically bind to such antigen. If a peptide is used, such peptides are considered particularly useful consisting of a t least eight or more, preferably eight to about forty consecutive amino acids of the p53 proline rich domain. Such antibody can enable us to isolated proteins with motifs simitar to the P-X-X-P motif or the p53 proline rich domain. Such proteins may play a role in disease related processes.

This invention further covers a nucleic acid (DNA, RNA) comprising an isolated, preferably recombinant, nucleic acid (DNA, RNA) coding for a mutant of the invention, or a fragment of such a nucleic acid. In addition to being useful for the production of the above-mentioned recombinant proteins of the invention, such isolated nucleic acids may be useful as probes, thus e.g. readily enabling those skilled in the art to identify and/or isolate nucleic acid encoding p53, p53 mutants, or other proteins that have similar motifs. Furthermore, nucleic acid according to the invention is useful e.g. in a method for determining the presence of p53,or p53 mutants, said method comprising hybridizing the DNA (or RNA) encoding (or complementary to) a mutant of the invention to test sample nucleic acid and to determine the presence of p53 or a mutant of the invention. The invention also provides a method for amplifying a nucleic acid test sample comprising a nucleic acid polymerase (chain) reaction with nucleic acid (DNA or RNA) encoding (or complementary to) a p53 mutant. Such method may involve sets of oligonucleotides.

In particular, the invention provides an isolated DNA molecule encoding a before- mentioned protein of the invention, or a fragment of such DNA. Preferred are DNA molecules encoding mutants or peptides of the invention above designated as being preferred. By definition, such a DNA comprises a coding single-stranded DNA, a double- stranded DNA consisting of said coding DNA and complementary DNA thereto, or this complementary (single stranded) DNA itself.

Nucleic acids encoding a p53 tumor suppressor gene product are available within the art or can be obtained by producing a cDNA using the published sequences and standard methods within the art. Given the guidance provided herein, a nucleic acid of the invention is obtainable according to methods well known in the art. The present invention further relates to a process for the preparation of such nucleic acids. For example, a DNA of the invention is obtainable by chemical synthesis, by recombinant DNA technology or by polymerase chain reaction (PCR), or any combination of these methods. A suitable method for preparing a nucleic acid of the invention may e.g. comprise the synthesis of a number of oligonucleotides, their use for amplification of DNA by PCR methods, and their splicing to give the desired DNA sequence.

Nucleic acids of the invention can be incorporated into vectors for further manipulation. Such vectors are also provided herein. Specifically, the invention concerns a recombinant DNA which is a hybrid vector comprising at least one of the above mentioned DNAs of the invention, particularly such DNA designated as being preferred.

The hybrid vectors of the invention comprise an origin of replication or an autonomously replicating sequence, one or more dominant marker sequences and, optionally, expression control sequences, signal sequences and additional restriction sites.

Preferably, a hybrid vector of the invention comprises an above described nucleic acid insert operably linked to an expression control sequence, in particular those described hereinafter.

Vectors typically perform two functions in collaboration with compatible host cells. One function is to facilitate the cloning of a nucleic acid that encodes a protein of the invention, i.e. to produce usable quantities of the nucleic acid (cloning vectors). The other function is to provide for replication and expression of the gene constructs in a suitable host, either by maintenance as an extrachromosomal element or by integration into the host chromosome (expression vectors). A cioning vector comprises the DNAs as described above, an origin of replication or an autonomously replicating sequence, selectable marker sequences, and optionally, signal sequences and additional restriction sites. An expression vector additionally comprises expression control sequences essential for the transcription and translation of the DNA of the invention. Thus an expression vector refers to a recombinant DNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into a suitable host cell, results in expression of the cloned DNA. Suitable expression vectors are well known in the art and include those that are replicable in eukaryotic and/or prokaryotic cells.

Most expression vectors are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even tnough it is not capabie of replicating independently of the host cell chromosome. DNA may aiso be amplified by insertion into the host genome. DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.

Advantageously, expression and cloning vectors contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.

Since the amplification of the vectors is conveniently done in E. coli. an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript vector or a pUC plasmid.

Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up p53 mutant nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes confering resistance to G418 or hygromycin. The mammalian cell transfectants are placed under selection pressure which only those transfectants are uniquely adapted to survive which have taken up and are expressing the marker.

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the p53 mutant nucleic acid. Such promoter may be inducible or constitutive. The promoters are operably linked to DNA encoding the p53 mutant of the invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector.

Promoters suitable for use with prokaryotic hosts include, for example, the β- lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding a mutant of the invention, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine- Delgarno sequence operably linked to the DNA encoding encoding a mutant of the invention. P53 mutant nucleic acid transcription from vectors in mammalian host cells may be controlled by a promoter compatible with the host cell systems, e.g. a promoter derived from the genome of a virus. Suitable plasmids for expression of a mutant of the invention in eukaryotic host cells, particularly mammalian cells, are vectors containing e.g. the promoter of cytomegalovirus (CMV), RSV, SV40 virus or MMTV LTR.

Transcription of a DNA encoding a protein according to the invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector.

The various DNA segments of the vector DNA are operatively linked, i.e. they are contiguous and placed into a functional relationship to each other employing conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a manner known in the art. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function of the desired mutant protein are known to those skilled in the art. Nuecleic acid presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), in situ hybridization, using an appropriately labelled probe based on a p53 nucleic acid sequence, binding assays, immunodetection and functional assays.

The invention further provides host cells capable of producing a mutant of the invention and containing heterologous (foreign) DNA encoding said mutant.

The nucleic acids of the invention can be expressed in a wide variety of host cells, e.g. those mentioned above, that are transfected with an appropriate expression vector. A protein of the invention may also be expressed as a fusion protein. Recombinant cells can then be cultured under conditions whereby the protein(s) or fusion protein encoded by the DNA of the invention is (are) expressed.

Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-prositive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5α and HB 101 , or Bacilli. Further host cells suitable for p53 mutant encoding vectors include eukaryotic microbes, such as filamentous fungi or yeast, e.g. Saccharomyces cerevisiae. Higher eukaryotic cells include insect, amphebian and vertebrate cells. The host cells referred to herein include cells in culture as well as cells that are within a host animal. DNA may be stably incorporated into the cells or may be transiently expressed employing conventional methods. While the DNA provided herein may be expressed in any suitable host cell, e.g. a host cell referred to above, preferred for expression of DNA encoding a mutant of the invention are systems suitable for expression in higher eukaryotes, particularly insect and mammalian expression systems, including commercially available systems and other systems known to those of skill in the art.

For example, a mutant according to the invention may advantageously be expressed in insect cell systems. Insect cells suitable for use in the method of the invention include, in principle, any lepidopteran cell which is capable of being transformed with an expression vector and expressing heteroiogous proteins encoded thereby. In particular, use of the Sf cell lines, such as the Spodoptera frugiperda cell line IPBL-SF-21 AE (Vaughn et al., (1977) In Vitro, 13, 213-217) is preferred. The derivative cell line Sf9 is particularly preferred. However, other cell lines, such as Tricoplusia ni 368 (Kurstack and Marmorosch, (1976) Invertebrate Tissue Culture Applications in Medicine, Biology and Agriculture. Academic Press, New York, USA) may be employed. These cell lines, as well as other insect cell lines suitable for use in the invention, are commercially available (e.g. from Stratagene, La Jolla, CA, USA).

As well as expression in insect cells in culture, the invention also comprises the expression of heteroiogous proteins in whole insect organisms. The use of virus vectors such as baculovirus allows infection of entire insects, which are in some ways easier to grow than cultured cells as they have fewer requirements for special growth conditions. Large insects, such as silk moths, provide a high yield of heteroiogous protein. The protein can be extracted from the insects according to conventional extraction techniques. It is also preferred to produce a mutant according to the invention using mammalian expression systems. Suitable host cells include cell lines expressing endogenous wildtype p53, such as fibroblasts, e.g GM11 (normal human fibroblast) and tumor cell lines, e g. U2- OS (osteosarcoma), as well as cell lines which do not have endogenous p53 protein, such as H1299, SAOS-2 10(3) and 10(1) cell lines and permanent cell lines which express a mutant of the invention at rather physiological concentrations and in a transcriptionally active form when shifted to 32°C for the case of a temperature sensitive p53 mutant. Such cell lines are e.g. publicly available, e.g. from the American Type Culture Collection (ATCC), or given out widely by the laboratory of Prof. Arnold J. Lev e, Princeton University, U.S.A

Stably transfected cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency. To produce such stably or transiently transfected cells, the cells should be transfected with a sufficient amount of p53 mutant-encoding nucleic acid to form protein of the invention. The precise amounts of DNA encoding a mutant of the invention may be empirically determined and optimized for a particular cell and assay.

Host cells are transfected or transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Heteroiogous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heteroiogous DNA by the calcium phosphate coprecipitation technique, by electroporation or lipofectin- mediated Successful transfection is generally recognized when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used (see, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).

A DNA of the invention may also be expressed in non-human transgenic animals, particularly transgenic warm-blooded animals such as mice. Methods for producing transgenic animals, including mice, rats, rabbits, sheep and pigs, are known in the art and are disclosed, for example by Hammer et al. ((1985) Nature 315, 680-683). An expression unit including a DNA of the invention coding for a mutant of the invention together with appropriately positioned expression control sequences, is introduced into pronuclei of fertilized eggs. Introduction may be achieved, e.g. by microinjection. Integration of the injected DNA is detected, e.g. by blot analysis of DNA from suitable tissue samples. It is preferred that the introduced DNA be incorporated into the germ line of the animal so that it is passed to the animal's progeny.

Replacing wild-type p53 into a transgenic mouse is most commonly lethal. To breed mice that express only a mutant of the invention, e.g. the p53dl(62-91) mutant, the respective transgene is crossed into a p53 null-mouse background. Study of such animal provides insights into the importance of the p53 proline rich domain in vivo.

The preferred p53 mutants, or cells producing such mutants, provided by the instant invention display all of the following properties:

- they may be stably expressed in tumor cells, such as H1299;

- they exhibit p53-mediated transcriptional activation comparable to or better than the wildtype protein;

- they respond to DNA damage comparable to wild-type p53;

- despite displaying wild-type transactivation properties, they have lost the wild-type p53 growth suppression phenotype (either G1 arrest or apoptosis);

- the growth suppression defect cannot be ascribed to either a failure to bind DNA or to activate transcription;

- they are compromised in their ability to signal for growth suppression in tumor cells; as such, they fail to regulate the cell cycle or induce apoptosis properly;

- based on their immunoreactivity with conformation-specific monoclonal antibodies, such as pAb421 , they display the conformation characteristic of wild-type p53.

The above-mentioned properties may be detected, and optionally quantified, in vitro or in vivo according to methods well-known to those skilled in the art, e.g. using the assays decribed in more detail in the Examples. Briefly, the ability of a mutant of the invention to suppress the growth of tumor cells may be determined in culture by a colony formation assay. Such assay enables growth suppresion in its most general sense to be measured as it scores for either p53-dependent growth arrest and/or apoptosis. (Lack of) Interaction with a proline rich domain may be demonstrated in a conventional binding assay in vitro or by co-immunoprecipitation in vivo. Apoptotic phenomenons can be investigated using the TUN(N)EL staining procedure with single cells. The transactivation potential may be determined according to the CAT assay protocols described in G.P. Zambetti et al, Genes & Develop. 6, 1143-1152 (1992) incorporated herein by reference. Such assay is suitable to determine the capacitiy of a p53 mutant to activate expression of a reporter under the regulation of a p53-response element. Induction of DNA damage is achievable by irradiation.

The invention also relates to a method of screening compounds or mixtures of compounds which are potential modulators of p53 direct signaling activity comprising the steps of (a) preparing a test system comprising a p53 protein capable of direct signaling; (b) exposing the test system to the compound or mixture of compounds; (c) identifying the compound or mixture of compounds which causes modulation of p53 direct signaling activity as measured by the test system. By comparing cells with wild-type p53 and the same cells with said proline rich mutant p53 of the invention for compounds that activate wild type but not mutant p53, one can search for compounds that induce an activity that acts on wild-type p53. In particular, the present invention provides a method for identification or design of a compound that is capable of modulating p53-mediated direct signaling function, said method comprising contacting wildtype p53, or a functional equivalent thereof, with a compound whose ability to modulate p53 direct signaling function is sought to be investigated and determining the change in p53 direct signaling function caused by the compound. Determination of the change in p53 direct signaling activity involves comparison to a suitable control, comprising e.g. contacting the candidate compound to a p53 mutant of the invention. A component which modulates p53 direct signaling acitivity includes a compound or signal that is capable of altering the response pathway mediated by the p53 proline rich domain within a cell (as compared e.g. to the absence of said domain). Modulation of p53 direct signaling function refers to modulation of the biological activities mediated via the p53 proline rich domain, particularly binding affinity and/or kinetics to a SH-3 domain, transcription-independent growth suppression and transcription-independent apoptosis. As mentioned above, assays for determining a change In any of these properties are well-known in the art. Such assays enable identification of compounds or agents which induce or enhance, or inhibit p53 direct signaling, e.g. by interacting with, particularly binding to, the proline rich domain of p53. In a preferred embodiment, the method aims at the identification of compounds which modulate p53- mediated signaling function without involving p53-mediated transcriptional activation. The instant invention also relates to a method for inducing in a cell p53-mediated apoptosis by activating a p53 function not dependent upon p53-medιated transcription, such as p21 induced growth arrest.

The invention further provides a method for the treatment of uncontrolled cell growth comprising modulating cell cycle regulatory proteins by introducing into said cell a peptide of the invention, or a compound which mimics such peptide.

Furthermore, the invention provides a method for treatment of a neurodegenerative or proliferative disease comprising introducing into a mammalian cell, particularly a human neuronal ceil or tumor cell, in which cell death cell cycle arrest is aberrant, a nucleic acid encoding a p53 mutant or peptide of the invention, or a compound that mimics such peptide.

For treatment, the compounds according to the present invention may be applied in tne form of a pharmaceutical composition, comprising a pharmaceutically effective amount of tne respective compound.

The following Examples serve to illustrate the present invention, but should not be construed as a limitation thereof. The invention particularly relates to the specific embodiments (e.g. the proteins, peptides nucleic acids, vectores, host cells, methods for their preparation, assays and uses thereof) as described in these Examples.

Examples

Example 1 : Construction of deletion mutant p53 dl(62-91)

Tne deletion mutant dl(62-91) is constructed by using PCR to independently amplify cDNAs encoding ammo acids 1-62 and amino acids 92-393 of human p53. The full length p53 cDNA cloned in the vector Bluescript KS (Stratagene) (J. Lin, J. Chen, B. Elenbaas, A. J.

Levine, Genes & Develop. 8, 1235-1246 (1994)) is used as a template. Primer pairs include the T3 forward primer (Bluescript sequence) and the reverse primer

5' CCGGATCCGGACCTGGGTCTTC 3' (p53 sequence with a 5'BamHI site; SEQ ID NO:1) for am o acids 1-62 and the forward primer 5' CCGGATCCCCTGTCATCTTCTG 3' (p53 sequence with a 5' BamHI site; SEQ ID NO:2) and the T7 reverse primer (Bluescript sequence) for amino acids 92-393. Both PCR products are directly cloned into the TA vector (Invitrogen) and subsequently joined through ligation at the internal BamHI sites. Example 2: Transactivation Assay

The deletion mutant dl(62-91) is placed under the control of the CMV promoter in the expression vector pRC-CMV (Invitrogen) so as to express the mutant protein in cells. This cDNA is transfected into cells without any endogenous p53 (H1299) along with a p53- responsive reporter construct WAF1-CAT, containing the regulatory elements derived from the p21 gene. cDNAs encoding for wildtype p53, the double point mutant L22Q-W23S (22,23), the tumor derived mutant R175H (175) are also cloned into the mammalian expression vector pRC-CMV for transactivation assays. The cDNA for the human wild-type p53 protein serves as a positive control, while the p53 double point mutant 22,23 (containing two ammo acid substitutions in the transcriptional activation domain) and the tumor derived mutant R175H (with a single ammo acid substitution in the DNA binding domain) serve as negative controls (J. Lm, J. Chen, B. Elenbaas, A. J. Levine, Genes & Develop 8, 1235-1246 (1994)). Cells are co-transfected with 100 ng of p53 expression Dlasmid and 1 ug of the p53-responsιve reporter WAF1-AT using standard calcium phosphate procedures. Cells are harvested approximately 72 hours post-transfection. Cell lines and transfection and CAT assay protocols are as described in G. P. Zambetti, J., Bargonetti, K Walker, C Prives, A. J. Levine, Genes & Develop. 6, 1 143-1 152 (1992), which is herein included by reference. The CAT assay testing the transactivation potential of tne aforementioned plasmids is each done in duplicate. Data are quantitated using a phosphoπmager and displayed graphically as the fold activation of the reporter over background

Both the 22,23 and R175H mutants demonstrate severe defects in transactivation, confirming the requirement for both intact DNA binding and transactivation domains. In contrast, the dl(62-91) mutant activates the expression of WAF1-CAT to a level equivalent of wild-type p53. These results are recapitulated with the p53-responsive reporter COSX1- CAT which is derived from the MDM2 gene demonstrating the generality of the reporter sequences. Moreover, when assayed in the p53-null cell lines SAOS-2 (human osteo- sarcoma) and (10)1 (immortalized murine), the activity of the dl(62-91) mutant is also indistinguishable from that of the wild-type protein showing an independence of the cell type used. These observations indicate that p53's proline rich domain is dispensable for transactivation and establish dl(62-91) as an unusual p53 mutant in having sustained a dramatic structural change without a consequent loss of transactivation potential. Example 3: Growth suppression assay

Based on the idea that transcriptional activation is correlated with p53mediated growth suppression, the activity of the dl(62-91) mutant in a growth suppression assay is tested. The ability of the dl(62-91) mutant to suppress the growth of tumor cells in culture is determined by a colony formation assay. This assay enables growth suppression in its most general sense to be measured as it scores for either p53-dependent growth arrest and/or apoptosis. Plasmids containing wild-type p53 or the dl(62-91) mutant in as with a G418- resistance marker are transfected into the H1299 line (null for p53) and geneticin-resistant colonies are scored. Controls include the transactivation mutant 22,23 and the DNA binding mutant R175H.

H1299 cells are transfected with 5 μg of the empty pRC/CMV vector or the same vector containing the above indicated p53 or p53 mutant cDNA by standard calcium phosphate procedures. Cells are harvested and replated at 2.5 x 10⁴ cells per plate approximately 36 hours post-transfection. 24 hours later, cells are plated under drug selection (0.8mg/ml geneticin) and drug-resistant colonies are scored approximately three weeks later. Average ratios are obtained from four independent experiments.

As a potent suppressor of tumor cell growth in culture, the wild-type p53 plasmid reduces the number of drug-resistant colonies tenfold, yielding an average ratio of 0.1 colonies relative to the empty vector control. Previous experiments have demonstrated that these colonies either contain no p53 or a mutant p53 plasmid (C. A. Finiay, P. W. Hinds, A. J. Levine, Cell 57, 1083-1093 (1989)). The R175H mutant completely looses the tumor suppressor phenotype (average ratio of 1.03 colonies relative to the empty vector control) while the transactivation mutant, 22,23, is also severely impaired in its ability to stop cell division or kill cells (average ratio of 0.59 colonies relative to the empty vector control). The dl(62-91) mutant is also greatly compromised for growth suppression, achieving less than a two-fold decrease in colony number (average ratio of 0.57 colonies relative to the empty vector control). Therefore, the removal of the proline rich domain from the p53 protein reduces the efficiency of growth suppression in culture approximately five-fold when compared to the wildtype p53 cDNA. These same results can be repeated with other cell lines (SAOS-2). Because the dl(62-91) mutant maintains its activity as a transactivator, these results indicate that p53-mediated growth suppression can be uncoupled from transcriptional activation. That these two activities are, in fact, separable, has precedence in the observations that p53 mutants defective for transcriptional activation still maintain activity in assays measuring suppression of oncogene-mediated transformation (T Unger, J A Mietz, M. Scheffner, C. Yee, P M. Howley, Mol. & Cell Biol 13, 5186-5194 (1993), T Crook, N J Marston, E. A. Sara, K H Vousden, Ce//79, 817 (1994)) or apoptosis (A J Wagner, J M. Kokontis, N Hay, Genes & Devel. 8, 2817-2830 (1994); Y. Haupt, W. S. Alexander, G. Bam, S. P. Klmker, J M. Adams, Ce//65, 753-763 (1991 )). However, this is the first report of a p53 mutant which despite displaying wild-type transactivation properties nas lost the wild-type p53 growth suppression phenotype These data suggest that the p53's proline rich domain mediates an activity critical for the effective transmission of p53 anti-proliferative signals

Example 4 Expression of dl(62-91) and further characterization of the mutant To confirm that the defect in growth suppression manifest by the dl(62-91) mutant does not merely reflect an instability of the protein in tumor cells, several drug-resistant H1299 colonies are expanded into clonal cell lines and tested for expression of p53 protein For stable expression in tumor cell lines, H1299 cells are transfected with 5 μg of the plasmid dl(62-91)/pRC-CMV and αrug-resistant colonies are selected and expanded in media supplemented with 0 8mg/ml geneticm (BRL) Three independent cell lines (designated dipro I, dlpro.2, and dipro 3) as well as the parental line H1299 are analyzed for p53 protein expression by immunoprecipitation/Western analysis. Cell lysates are immunoprecipitated with the p53-specιfιc monoclonal antibody, pAb421 , or the negative control antibody, pAb419, as previously described (J Momand G P Zambetti, D. C. Olson, D George, A J Levine, Cell 69, 1237-1245 (1992)). Immunocomplexes are resolved in a 10% SDS gel and transferred to a membrane which is probed with the p53-specifιc antibody, pAb421 Protein A-conjugated peroxidase (Boehπnger Mannheim) and ECL luminescence (Amersham) is used to detect bound antibody, according to manufacturer's directions.

In contrast to the H1299 cell line, each of the three dipro lines expresses a protein reacting with the p53-specifιc monoclonal antibody, pAb421 but not the negative control antibody, pAb419 which does not bind to p53 The three dipro lines are heterogeneous with regard to the expression level of the p53 transgene, but the levels detected are approximately equal to cell lines with wild-type p53 protein Because of the fact that tumor cells are not able to tolerate the stable re troduction of wild-type p53, the creation of tumor lines stably expressing the dl(62-91) mutant strongly suggests that the removal of the proline rich αomain impairs some aspect of growth suppression or apoptosis.

Metabohcally labeled cell lysates (J. Momand. G. P. Zambetti, D. C. Olson, D. George. A. J. Levine, Cell 69, 1237-1245 (1992)) generated from two dipro lines (dipro.1 and dlpro.3), the parental line H1299, and two human cell lines expressing endogenous wildtype p53, GM1 1 (normal human fibroblast) and U2-OS (osteosarcoma) are immunoprecipitated with the p53- specific antibody, pAb421 , or negative control antibody, pAb419. Complexes are resolved in a 10% SDS gel Radiolabeled proteins are detected by exposure to X-ray film. Comparison of the levels of dl(62-91) expression with those of wild-type p53 in cell lines expressing endogenous p53 by immunoorecipitation of radiolabeled cell lysates shows that the dl (62-91 ) protein migrates faster than the full length p53 protein, consistent with the αecrease in molecular weight resulting from the deletion of the proline rich domain Importantly, the levels of dl(62-91) protein are very similar to the levels of wildtype p53 expressed in the GM11 primary human fibroblasts and the tumor line U2-OS This suggests that the removal of ammo acids 62-91 does not dramatically alter the short half-life of the wild-type p53 p^roteιn, in marked contrast to ammo acid substitutions in the DNA binding doma Moreover, based on its immunreactivity with conformation-specific monoclonal antibodies the dl(62-91) protein maintains a conformation characteristic of wild-type p53 Therefore the dl(62-91) mutant represents a novel class of p53 mutants for which a growth suppression defect cannot be ascribed to either a failure to bind DNA or activate transcription

One possibility to account for the unusual fact that the dl(62-91) protein is tolerated by tumor cells is that it has become functionally inactivated during the establishment of stable eel! lines This possibility may be tested by demonstrating that the dipro lines express an endogenous activity capable of activating transcription in a p53-dependent manner. Initially, this is approached by determining whether the dipro lines are capable of transactivating a transiently transfected p53-responsιve reporter construct.

To analyze the ability of stably expressed dl(62-91) to activate transcription, the three dipro ^lines (dipro 1 , dlpro.2, and dlpro.3) and the parental line H1299 are transfected with the p53-responsιve reporter WAF1-CAT or the negative control reporter Gal4-CAT in the presence or absence of the human MDM2 expression vector, HDM2 (J. Momand, G. P. Zambetti, D. C. Olson, D George, A. J Levine, Cell 69, 1237-1245 (1992)). In addition, the dl (62-91) expression plasmid is included in designated H1299 transfections. The level of reporter activation is determined 48 hours post-transfection. CAT assays are performed as previously described (G. P. Zambetti, J. Bargonetti, K. Walker, C. Prives, A. J. Levine, Genes & Develop. 6, 1143-1 152 (1992)).

Each of the dipro lines is able to transactivate the WAF1 -CAT reporter, but not the Gal4- CAT reporter. The parental line, in contrast, expresses this activity only when a plasmid containing the dl(62-91) cDNA is included in the transfection. These results indicate that stable expression of the dl(62-91) mutant confers upon cells the capacity to activate expression of a reporter under the regulation of a p53-response element. That this activity is directly p53-dependent is supported by the fact that it is suppressed by co-transfection of the human MDM2 protein, a known negative regulator of p53's transactivation function (J. Momand; G. P. Zambetti, D. C. Olson, D. George, A. J. Levine, Cell 69, 1237-1245 (1992)).

To further confirm that the dl(62-91 ) protein stably expressed in the tumor cells maintains the capacity to activate transcription, the steady-state protein levels of p21 , a known p53 target gene, are compared between parental the H1299 line and its dipro derivatives. To compare the steady-state levels of p21 protein in the dipro.1 and dlpro.3 lines to that in the parental H1299 line, cell lysates are immunoprecipitated with antisera specific for human p21 or pre-immune sera. Complexes are resolved in a 10% SDS gel and transferred to a nylon membrane. The membrane is probed with the same antisera to p21 and bound antibody is detected with ¹²⁵l-conjugated to protein A. Data are quantitated using phosphorimager analysis (Molecular Dynamics software). Quantitation of the data reveals that the dipro.1 line produces 4-fold more p21 as compared to the parental line, whereas the dlpro.3 lines produces 7-fold more. Taken together, these results suggest that p53 transcriptional inactivation is not occurring in these stably transfected dl (62-91) tumor cells.

Example 5: UV induction of dlpro(62-91)

There are several possible ways in which the proline rich domain of p53 can mediate the flow of growth-inhibitory information. For instance, it can participate in the reception of upstream alarm signal which lead to activation of the p53 protein; alternatively, it can function in the transmission of downstream signals which alter cell growth.

To address the possibility that the proline rich domain of p53 can mediate the flow of growth-inhibitory information by participating in the reception of upstream alarm signal which leads to activation of p53 protein, it is determined whether DNA damage can induce the levels of dl(62-91) protein stably expressed in the tumor lines The significant increase in p53 protein levels in response to DNA damaging agents is due at least in part to a post- translational stabilization of the protein. The cell line dlpro.3 is irradiated with a high dose of UV light (20J/m²) and the steady-state levels of p53 protein are determined at 0, 5, 9, and 12 hours post irradiation by immunoprecipitation followed by Western analysis using the pan-specific antibody, pAb421. As a control, the induction of wild-type p53 is monitored in the immortalized murme cell line (12)1 which expresses endogenous wild-type p53 (D Harvey, A. J. Levine, Genes & Develop. 5, 2375-2385 (1991)). Cell lysates are immunoprecipitated with the p53specifιc monoclonal antibody, pAb421 , or negative control antibody, pAb419. Immunocomplexes are resolved in a 10% SDS gel and transferred to a nylon membrane. The membrane is probed with the p53-specifιc antibody pAb421 and bound antibody is detected with ^1Z5l-conjugated protein A. Data are quantitated using phosphoπmager analysis (Molecular Dynamics).

The results show that wild-type p53 demonstrates a characteristic increase in concentration after UV treatment. Quantitation of the data indicates that the levels of wild-type p53 protein increase approximately six-fold by five hours and another two-fold by nine hours post- treatment Under identical experimental conditions, the levels of the dl(62-91) protein increase 20-fold by five hours and another 2-fold by nine hours post-treatment. These results provide clear evidence that p53's upstream regulation, i e. communication with damaged DNA intermediates, is intact in the dlpro.3 line.

The human p53 protein has been divided into four domains: 1) a transcriptional activation domain that contacts several components of TFIID residues (1-40), 2) a sequence specific DNA binding domain (residues 120-290), 3) a tetramenzation domain (residues 310-360), and 4) a domain that recognizes and binds to damage DNA (residues 364-390) (S. Lee, B. Elenbaas, A. J. Levine, J. Griffith, Cell 81, 1-20 (1995)). The instant invention discloses a new fifth domain, localized between aa residues 61 -94 containing a putative proline rich signaling domain. Deletion of this domain from the p53 protein leaves a normal p53 protein for transcriptional activation and response to DNA damage. The mutant p53 protein (p53dl (62-91)) however, is severely compromised in its ability to signal for growth suppression in tumor cells. As such, it fails to either regulate the cell cycle or induce apoptosis in these cells In H1299 cells expressing the p53dl(62-91) mutant protein, p21 levels are elevated and the cells do grow slower (generation time is longer) than H1299 celis without any p53 protein. This suggests that the proline rich domain of p53 localized between residues 62-91 is involved in signaling for programmed cell death.

The experiments presented here demonstrate two requirements for p53-mediated growth suppression in H1299 cells: 1) the need for a p53-mediated transcriptional step which is abrogated by p53 mutants 22,23 and 175 in the transactivating or DNA binding domains, and 2) the need for direct signaling via the 62-91 proline rich domain. Defects in either of these domains reduce cell suppression of growth, i.e. colony formation, by p53. It is possible that direct signaling by p53dl(62-91) domain is accomplished when the protein is bound to DNA.

is believed that the p53 proline rich domain possibly signals via its P-X-X-P sequences contacting an SH3 domain of another protein. This may occur after DNA damage when increased levels of p53 are present (downstream). The c-abl protein is a candidate target with an SH3 binding domain. It is activated after DNA damage (S. Kharbanda, et al., Nature 376, 785-788 (1995)), binds to p53 under some circumstances (A. Goga, et al., Oncogene . ..791 (1995)), ana is involved in growth regulation (C. L. Sawyers, J. McLaughlin, A. Goga, M. Havlik, O Witte. Ce//77, 121 -131 (1994)). Peptides containing the p53 61 -94 ammo aciα sequences bind to the SH3 binding domain of c-abl in vitro. This pathway might be critical in regulating cellular responses to oncogene activation or DNA damage. Because the P-X-X-P signals in human p53 are redunαant (5 copies), this would be a difficult target for selection of mutations in cancers even though it is essential to the tumor suppressor function of p53.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Trustees of Princeton University

(B) STREET: 5 New South Building

(C) CITY: Princeton NJ

(E) COUNTRY: USA

(F) POSTAL CODE (ZIP): 08544

(G) TELEPHONE: 001 609 258 3097 (K) TELEFAX: 001 609 258 1159

(ii) TITLE OF INVENTION: P53 Mutant

(iii) NUMBER OF SEQUENCES: 4

fiv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) ΞTRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic oligonucleotide"

(iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CCGGATCCGG ACCTGGGTCT TC 22

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic oligonucleotide"

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

CCGGATCCCC TGTCATCTTC TG 22

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: s.ingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:1..102

(D) OTHER INFORMATION: /product^*= "peptide consisting of amino acids 61 to 94 of human p53"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GAT GAA GCT CCC AGA ATG CCA GAG GCT GCT CCC CCC GTG GCC CCT GCA 48 Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala 1 5 10 15

CCA GCA GCT CCT ACA CCG GCG GCC CCT GCA CCA GCC CCC TCC TGG CCC 96 Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro 20 25 30

CTG TCA 102

Leu Ser

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 .amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala 1 5 10 15

Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro 20 25 30

Leu Ser

Claims

1. A protein derivable from p53 which lacks all or part of the p53 proline rich domain and is therefore essentially unable to interact via said domain with another protein or chemical moiety.

2. A protein according to claim 1 which is derivable from human wild-type p53.

3. A protein according to claim 1 which lacks from at least two to ail P-X-X-P motifs present the proline rich domain of wild-type p53.

4. A protein according to claim 1 which lacks essentially the complete proline rich domain ana is designated dl(62-91).

5 A method of preparing a protein according to claim 1 comprising chemical synthesis or recombinant DNA techniques.

6 A nucleic acid encoding a Drotein according to claim 1.

7 A replicable nucleic acid vector comprising a coding sequence consisting of a nucleic acid according to claim 6 operable linked to suitable control sequences.

8. A nost cell transformed with a vector according to claim 7.

9. A Deptide which is capable of selectively binding to a SH3 domain and comprises all or part of the p53 proline rich domain.

10. A peptide according to claim 9 comprising the amino acid sequence set forth in SEQ ID NO:4.

11 . An o gonucleotide encoding a peptide according to claim 9.

12. A method for inducing p53-mediated apoptosis, in a cell in the absence or presence of p53 mediated transcriptional activation, comprising inducing or enhancing the direct signalling pathway mediated by the p53 proline rich domain.

13. A method for inhibiting uncontrolled cell growth or cell degeneration comprising modulating cell regulatory proteins by introducing into said cell a peptide according to claim 9.

14. A method for treatment of a neurodegenerative or proliferative disease comprising introducing into a mammalian cell a nucleic acid encoding a protein according to claim 1.

15. A method for treatment of a neurodegenerative or proliferative disease comprising introducing into a mammalian cell a nucleic acid encoding a peptide according to claim 9, or a suitable peptidomimetic.

16. A method of identifying a compound or a mixture of compounds which is capable of activating the cellular pathway which induces a molecule that interacts with the proline rich domain of p53, said method comprising exposing said compound or mixture to a protein according to claim 1 and determining the ability of said compound or mixture to activate the protein according to claim 1.

17. A fusion protein containing the proline rich domain of p53 whose overexpression in cells blocks p53-mediated activities.