Abstract:
The transcription factor and tumor suppressor protein p53 is inactivated in many human cancers. Approximately forty percent of cancers carry large amounts of mutated full-length p53 protein with one of over 900 reported single amino acid changes in the p53 core domain that recognizes p53 DNA binding sites. The ability to restore function to these inactive p53 proteins would dramatically improve cancer therapy. We show that this goal is achievable. Using genetic strategies and p53 assays in the yeast  S. cerevisiae  and mammalian cells, we identify suppressor mutations that, in the context of the some protein, restore function to some of the most common p53 cancer mutants tested. Crystallographic studies of this general suppressor motif eludicate the structural basis of restoring integrity to the p53 core domain and lead to small compounds that stabilize p53 cancer mutants.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application claims the benefit under 35 U.S. C. § 119(e) to U.S. provisional application 60/348,394 filed Jan. 16, 2002, the disclosure of which is incorporated by reference in its entirety. 
     
    
       [[0002]]     This invention was made with government support under grant number CA 81511 awarded by the National Institutes of Health/National Cancer Institute. The government has certain rights to the invention. 
     
    
     FIELD OF THE INVENTION  
       [0003]     The invention is related to the field of cancer therapy. More particularly, it is related to intragenic suppressor motifs in the p53 tumor suppressor protein that restore function to common mutant p53 proteins of cancer patients.  
       BACKGROUND OF THE INVENTION  
       [0004]     p53 inactivation and cancer. The tumor suppressor gene p53 is of central importance for the genetic stability of human cells (Donehower and Bradley, 1993; Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997). The p53 protein is active as a homo-tetramer and exerts its tumor suppressor function mainly as a transcription factor that induces G1 and G2 cell cycle arrest and/or apoptosis (Donehower and Bradley, 1993; Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997; Hermeking et al., 1998). The p53-mediated G1 arrest is its best characterized activity and involves transcriptional activation of the downstream gene p21 WAF1/CIP1/SDI1  (Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997). Other downstream effector genes for p53-mediated G1 arrest may exist, since p21 −/−  mouse embryonic fibroblasts do not show complete abrogation of G1 arrest after DNA damage (Brugarolas et al., 1995; Deng et al., 1995). The G2/M block mediated by p53 involves, at least in part, induction of 14-3-3σ (Hermeking et al., 1998).  
         [0005]     The mechanisms for apoptosis induction and their relative importance remain less clear at present. In certain settings p53 clearly induces pro-apoptotic genes. These include BAX and Fas/APO1 (Miyashita and Reed, 1995; Owen-Schaub et al., 1995) neither of which, however, is an absolute requirement for p53-induced apoptosis (Knudson et al., 1995; Fuchs et al., 1997; Yin et al., 1997). Recently, many more genes have been identified that are induced directly or indirectly during p53-mediated apoptosis (Polyak et al., 1997; Wu et al., 1997; Yin et al., 1998), but the essential genes for p53-induced apoptosis still have to be determined. Transcriptional repression of anti-apoptotic genes, such as bcl-2, may play a role (Haldar et al., 1994; Miyashita et al., 1994) and other non-transcriptional mechanisms maybe important as well (Caelles et al., 1994; Wagner et al., 1994; Haupt et al., 1995; Wang et al., 1996; White, 1996).  
         [0006]     Several upstream signals activate p53. These include DNA damage, hypoxia and critically low ribonucleoside triphosphate pools (Kastan et al., 1991; Graeber et al., 1996; Linke et al., 1996). Once activated, p53 induces either cell cycle arrest or apoptosis, depending on several factors such as the amount of DNA damage, cell type and cellular milieu, e.g., presence or absence of growth factors (Donehower and Bradley, 1993; Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997).  
         [0007]     Cancer cells show decreased fidelity in replicating their DNA, often resulting in DNA damage, and tumor masses have inadequate neovascularization leading to ribonucleoside triphosphate or oxygen deprivation, all upstream signals that activate p53. In view of p53&#39;s capability to induce cell cycle arrest or apoptosis under these conditions it is not surprising that absent or significantly reduced activity of the tumor suppressor protein p53 is a characteristic of more than half of all human cancers (Hollstein et al., 1991; Harris and Hollstein, 1993; Greenblatt et al., 1994). In the majority of cancers, p53 inactivation is caused by missense mutations in one p53 allele, often with concomitant loss-of-heterozygosity (Michalovitz et al., 1991; Vogelstein and Kinzler, 1992; Donehower and Bradley, 1993; Levine, 1997). These mutations affect almost exclusively the core DNA-binding domain of p53 that is responsible for making contacts with p53 DNA-binding sites (Cho et al., 1994), while mutations in the N-terminal transactivation domain or the C-terminal tetramerization domain are extremely rare ( FIG. 1 ) (Beroud and Soussi, 1998; Cariello et al., 1998; Hainaut et al., 1998). Contrary to wild-type p53, p53 cancer mutants have a long half-life and accumulate to high levels in cancer cells (Donehower and Bradley, 1993; Lowe, 1995). This may be explained by their inability to activate the MDM-2 gene (Lane and Hall, 1997), since mdm-2 induces degradation of p53 via the ubiquitin pathway as part of a negative feedback loop (Haupt et al., 1997; Kubbutat et al., 1997). The unusually high frequency of p53 missense mutations in human cancers (as opposed to mutations resulting in truncated proteins) is explained by their dominant-negative effect that depends on the intact C-terminal tetramerization domain. The C-terminus allows p53 cancer mutants to form hetero-tetramers with wild-type p53 (Milner and Medcalf, 1991), thus reducing, or even abrogating, the activity of wild-type p53 protein (Michalovitz et al., 1991; Vogelstein and Kinzler, 1992; Hann, 1995; Brachmann et al., 1996; Ko and Prives, 1996). In addition, there is evidence that at least some of the same missense mutations may confer a gain-of-function (Gottlieb and Oren, 1996; Ko and Prives, 1996; Levine, 1997).  
         [0008]     p53 abnormalities and cancer therapy. Considering the activities of the p53 tumor suppressor protein, reconstitution of wild-type p53 activity to cancers would be of large therapeutic benefit, an idea that is supported by several lines of evidence from epidemiological, clinical and basic cancer research (Fisher, 1994; Lowe, 1995; Harris, 1996a).  
         [0009]     Several human malignancies that are usually diagnosed at a young age, such as testis cancer, pediatric acute lymphoblastic leukemia and Wilms tumor, can be successfully eradicated even at advanced stages. They all have in common that they carry wild-type p53 (Heimdal et al., 1993; Wada et al., 1993; Malkin et al., 1994). At the same time, subgroups of these malignancies with a poor prognosis, for example the anaplastic variant of Wilms tumor, commonly do carry p53 mutations (Bardeesy et al., 1995). Similarly, tumor types that are often resistant to conventional therapies and difficult to treat at advanced stages, such as lung, prostate, colorectal, breast, head and neck, pancreatic and gastric cancers, show a high frequency of p53 mutations (Hollstein et al., 1991; Fisher, 1994; Lowe, 1995; Harris, 1996a; Beroud and Soussi, 1998; Cariello et al., 1998; Hainaut et al., 1998).  
         [0010]     These findings have spurred great interest in exploring p53 as a predictive marker for response to therapy and for overall prognosis. The majority of cancer types have been evaluated to some extent, and the publications are too numerous to be summarized here. As an example, studies in breast, head and neck, lung and ovarian cancers have found a good correlation between p53 abnormalities and poor survival and poor response to therapy (Thor et al., 1992; Allred et al., 1993; Bergh et al., 1995; Rusch et al., 1995; Sauter et al., 1995; Righetti et al., 1996; Berns et al., 1998; Huang et al., 1998). The results are not always unequivocal, as some studies were unable to detect a statistically significant difference between cancers with and without functional p53 (Isola et al., 1992; Elledge et al., 1995). These discrepancies may be due to confounding factors. For example, a cancer with a poor prognosis because of degradation of p53 by overexpressed mdm-2 may be incorrectly scored as a cancer with functional p53 if the mdm-2 status of the cancer is not evaluated. In addition, the sample size of many studies was not large enough to make firm conclusions.  
         [0011]     Strong evidence for a central role of p53-mediated apoptosis in cancer therapy is provided by experiments in cell lines with and without functional p53. Comparison of wild-type and p53-deficient thymocytes established that p53 is required for radiation- and etoposide-induced apoptosis (Clarke et al., 1993; Lowe et al., 1993a). Similar experiments in adenovirus E1A transformed mouse embryo fibroblasts showed that apoptosis induced by radiation, 5-fluorouracil, etoposide and adriamycin also depends on functional p53 in these cells (Lowe et al., 1993b). These studies were extended into a mouse model where again only tumors with functional p53 showed good treatment responses to radiation and adriamycin, while p53-negative tumors were highly resistant to therapy and showed little evidence of apoptosis (Lowe et al., 1994). Results of the Developmental Therapeutics Program of the NCI impressively and independently confirmed these findings. An analysis of the cytostatic and cytotoxic effects of 123 compounds on 60 different human cancer cell lines showed a very good correlation between p53 mutations and resistance to many commonly used chemotherapeutic agents (O&#39;Connor et al., 1997; Weinstein et al., 1997). All these data do not necessarily indicate that functional p53 is absolutely essential for chemotherapy-induced apoptosis. In fact, chemotherapy drugs can kill cancer cells through p53-independent mechanisms (Kaufmann, 1989; Strasser et al., 1994; Bracey et al., 1995). The sum of the evidence, however, suggests that cancer agents are significantly more effective in the presence of p53 (Fisher, 1994; Lowe, 1995; Harris, 1996a).  
         [0012]     Based on the discussed studies and the general knowledge about p53, p53 and its pathways have been recognized as a prime target for developing new cancer therapies (Fisher, 1994; Gibbs and Oliff, 1994; Kinzler and Vogelstein, 1994; Lowe, 1995; Milner, 1995; Harris, 1996a). In particular, the high frequency of p53 mutations in cancers makes therapeutic strategies for restoring this tumor suppressor pathway highly desirable since a large number of patients could potentially benefit. It has been estimated that every year approximately 330,000 patients in the United States and 2.4 million patients worldwide are diagnosed with cancers that contain p53 mutations (Harris, 1996a, 1996b).  
         [0013]     Strategies to partially or completely restore wild-type p53 function to cancer cells. Restoration of wild-type p53 activity to cancer cells is the most direct way of making cancer cells more susceptible to apoptosis and can be pursued in two ways. The first strategy is to reintroduce wild-type p53, perhaps by gene therapy (Roth et al., 1996; Barinaga, 1997; Nielsen and Maneval, 1998), and does not rely on the p53 status of a given cancer. The current major challenge is efficient and selective targeting of wild-type p53 expression constructs to the cancerous cells (Nielsen and Maneval, 1998). A major drawback of this approach is that it may be less effective for cancers with vast amounts of a dominant-negative p53 cancer mutant. This strategy would be greatly aided by the availability of p53 proteins that are resistant to the dominant-negative effects of p53 cancer mutants and that are superior to wild-type p53 in inducing apoptosis, classes of p53 proteins that to date have not been described.  
         [0014]     The second strategy is only possible because of the unique pattern of p53 missense mutations in human cancers and aims at therapeutically exploiting the abundant p53 mutant protein found in many cancers. Since the resulting p53 cancer mutants are full-length proteins each with a structurally altered core domain, but an intact transactivation domain and an intact C-terminal tetramerization domain, one could restore wild-type activity to the p53 cancer mutants in these tumors (Gibbs and Oliff, 1994; Lowe, 1995; Milner, 1995; Harris, 1996a). This can be achieved in at least two ways. One attempt has been to interfere with the extreme C-terminal autoregulatory domain of p53 by using antibodies (Halazonetis and Kandil, 1993; Hupp et al., 1993; Abarzua et al., 1995; Niewolik et al., 1995) or peptides spanning part of this region (Hupp et al., 1995; Abarzua et al., 1996; Selivanova et al., 1997). This strategy presumably activates p53 cancer mutants by blocking the ability of the very C-terminus to fold back onto and inhibit the p53 core domain. It could succeed with p53 cancer mutants that retain residual activity and which only require additional activation to exceed the threshold required for biological effects. However, antibodies and peptides clearly cannot be delivered efficiently to cancer cells in patients (Selivanova et al., 1997). Small molecule compounds with similar effects could overcome this problem, but their design is currently not feasible since the exact structural basis of this negative autoregulation and of its neutralization by antibodies or peptides is not known due to lack of a crystal structure for the full-length p53 protein (Ko and Prives, 1996; Selivanova et al., 1997). In addition, this approach may activate mutant and wild-type p53 proteins indiscriminately, thus possibly causing significant side effects due to inappropriate wild-type p53-induced apoptosis in normal tissues.  
         [0015]     A more direct approach is to revert the effects of tumorigenic mutations on the structure and function of the p53 core domain itself by means of small molecules. This strategy is preferable since it is predicted to selectively stabilize p53 cancer mutants. It also holds the promise of restoring function to completely inactive p53 cancer mutants. Restoring the normal configuration to a p53 cancer mutant is considered more challenging than inhibiting the function of a protein by small molecules (Gibbs and Oliff, 1994). However, there are examples: small molecule compounds that bind the central cavity of the hemoglobin tetramer can act as allosteric effectors and stabilize the T state of hemoglobin over the R state (Abraham et al., 1992); and small molecule compounds that stabilize the transthyretin tetramer against dissociation can prevent amyloid fibril formation in vitro (Miroy et al., 1996). Furthermore, the technology of structure-based drug design is steadily advancing so that this challenge may be met (Bohacek et al., 1996; Marrone et al., 1997).  
         [0016]     p53 mutations and the p53 core DNA-binding domain. These considerations make it clear that a detailed understanding of the structural consequences of p53 cancer mutations on the p53 core domain is needed. More significantly, stabilizing mechanisms must be identified that can override the deleterious structural effects of p53 cancer mutations.  
         [0017]     The crystal structure of the wild-type p53 core domain has given enormous insight into how p53 interacts with its DNA-binding sites (Cho et al., 1994). The structures of the C-terminal tetramerization domain and of the N-terminal transactivation domain (complexed to mdm-2) have been determined as well (Clore et al., 1994; Jeffrey et al., 1995; Kussie et al., 1996). The structure of the full-length protein as a homo-tetramer, however, is solely based on computer modeling (Jeffrey et al., 1995) and suggests that the core domain functions as a separate entity that is connected to the other domains through flexible linkers. The core domain spans 191 amino acids and consists of a β sandwich that serves as the scaffold for two large loops (termed L2 and L3) and a loop-sheet-helix motif. The loops and the loop-sheet-helix motif form the DNA-binding surface of p53 and provide contacts to the DNA backbone and the edges of the bases ( FIG. 1A ). This structural organisation was considered unique until the recent discovery of p73 made it clear that p53 is actually part of a family of transcription factors (Jost et al., 1997; Kaghad et al., 1997). The vast majority of tumor-derived p53 missense mutations map to this core domain ( FIG. 1B ) and invariably result in the reduction or loss of DNA-binding. These cancer mutations are predicted to fall into two classes; one class of mutations maps to DNA-contacting residues and eliminates p53-DNA contacts (functional mutations); the other, larger class of mutations probably affects the structural integrity of the DNA-binding domain (structural mutations). These structural defects may range from small structural shifts to the global destabilization and unfolding of the p53 core domain. The most frequent p53 cancer mutations affect amino acids that are part of important structures of the p53 core domain, such as the L3 loop and the loop-sheet-helix motif that provide DNA contacts. However, the high frequency of a mutation does not predict how deleterious its effects on the structural integrity of the core domain are, since the frequency of these mutations is also determined by exogenous carcinogens and endogenous biological processes (Donehower and Bradley, 1993; Greenblatt et al., 1994).  
         [0018]     To date, our understanding of the structural consequences of p53 cancer mutations is limited to predictions using the structure of the wild-type p53 core domain, biochemical data (Cho et al., 1994) and experiments with monoclonal antibodies that recognize areas of the p53 core domain that are not accessible in the correctly folded state (Donehower and Bradley, 1993; Gottlieb and Oren, 1996; Levine, 1997). Similarly, very little is known about how the effects of cancer mutations can be overcome.  
         [0019]     There is a need in the art for the identification of p53 suppressors that are either resistant to the dominant-negative effects of p53 cancer mutants or are more efficient in inducing cell cycle arrest and/or apoptosis for the treatment of cancer.  
       BRIEF SUMMARY OF THE INVENTION  
       [0020]     In one embodiment of the invention a nucleic acid molecule encoding human p53 is provided. The nucleic acid has at least one intragenic suppressor mutation. The intragenic suppressor mutation is capable of suppressing a mutation of human p53 that occurs in human cancers when the intragenic suppressor and cancer mutations are present in a cis configuration. The intragenic suppressor mutation is selected from the group consisting of: T81S; A83V; P87R; Q100L; Q100R; Q104P; F113L; L114V; T118M; V122I; C124S; K139R; Q144L; W146R; Q165L; V172I; H178Y; S183T; A189V; F212L; E224G; S227P; S227T; D228N; D228A; D228E; C229W; T230S; T231I; I232V; H233R; H233Y; Y234F; N235K; N235S; Y236N; N239M; N239W; N239L; N239F; N239R; N239H; S240Q; S240T; S240R: D281G; E285G; E285K; E294G; G325R; E343V; E346G; D352G; and combinations thereof.  
         [0021]     In another embodiment of the invention a nucleic acid molecule encoding human 53 is provided. The nucleic acid has a set of at least one intragenic suppressor mutation. The set is capable of suppressing a mutation of human p53 that occurs in human cancers when the intragenic suppressor and cancer mutations are present in cis configuration. The set is selected from the group consisting of: F113L; L114V+T123P+V172I+A189V; T123P+A189V; S227P+N239Y; T118M+H168R; V122I+C124S+H168R; T123A+H168R; K139R+H168R+N239Y; H168R+T231I; T123A+S240R; H178Y+S240R; D281 G+E285G+G325R+E343V; E285K+D352G; E285K+E294G+E346G; T81S+A83V+S240R; P87R+Q100L+Q 104P+Q 144L+S240R; Q100R+W146R+S240R; Q165L+F212L+S240R; S183T+S240R; E224G+S240R; S240R; D228A+N235K+N239M; D228E+N235K+Y236N+N239L; I232V+H233R+Y234F+N235K+N239L; N235K+S240T; Y234F+N239L; N235K+N239R; H233Y+N239Y; N239F; N239Y+S240Q; H233Y+N235K+N239Y; N235K+N239Y; N235S+N239Y+S240N; D228N+N239Y; N235K+N239W; D228E+N239Y; N235K+S240N; N239W; T230S+N239Y; S227T+N235K+N239Y; H233R+N235S+N239R+S240R; N239R; N239R+S240R; C229W+N239R+S240R; N239L+S240R; N239F+S240R; I232V+N239H+S240R; D228E+C229W+N235K+N239Y+S240R; N239Y+S240R; and combinations thereof.  
         [0022]     In another embodiment of the invention a human p53 protein is provided. The protein has at least one intragenic suppressor mutation. The intragenic suppressor mutation is capable of suppressing a mutation of human p53 that occurs in human cancer when the intragenic suppressor and the cancer mutations are present in a cis configuration. The intragenic suppressor mutations is selected from the group consisting of: T81S; A83V; P87R; Q100L; Q100R; Q104P; F113L; L114V; T118M; V122I; C124S; K139R; Q144L; W146R; Q165L; V172I; H178Y; S183T; A189V; F212L; E224G; S227P; S227T; D228N; D228A; D228E; C229W; T230S; T231I; I232V; H233R; H233Y; Y234F; N235K; N235S; Y236N; N239M; N239W; N239L; N239F; N239R; N239H; S240Q; S240T; S240R; D281G; E285G; E285K; E294G; G325R; E343V; E346G; D352G; and combinations thereof.  
         [0023]     In another embodiment of the invention a human p53 protein is provided. The protein comprises a set of at least one intragenic suppressor mutation. The set is capable of suppressing a mutation of human p53 that occurs in human cancers when the mutation is present in cis configuration. The set is selected from the group consisting of: F113L; L114V+T123P+V172I+A189V; T123P+A189V; S227P+N239Y; T118M+H168R; V122I+C124S+H168R; T123A+H168R; K139R+H168R+N239Y; H168R+T231I; T123A+S240R; H178Y+S240R; D281G+E285G+G325R+E343V; B285K+D352G; E285K+E294G+E346G; T81S+A83V+S240R; P87R+Q100L+Q104P+Q144L+S240R; Q100R+W146R+S240R; Q165L+F212L+S240R; S183T+S240R; E224G+S240R; S240R; D228A+N235K+N239M; D228E+N235K+Y236N+N239L; I232V+H233R+Y234F+N235K+N239L; N235K+S240T; Y234F+N239L; N235K+N239R; H233Y+N239Y; N239F; N239Y+S240Q; H233Y+N235K+N239Y; N235K+N239Y; N235S+N239Y+S240N; D228N+N239Y; N235K+N239W; D228E+N239Y; N235K+S240N; N239W; T230S+N239Y; S227T+N235K+N239Y; H233R+N235S+N239R+S240R; N239R; N239R+S240R; C229W+N239R+S240R; N239L+S240R; N239F+S240R; I232V+N239H+S240R; D228E+C229W+N235K+N239Y+S240R; N239Y+S240R; and combinations thereof.  
         [0024]     In another embodiment of the invention a human p53 polypeptide is provided. The polypeptide has a portion of human p53 protein. The portion comprises residues 94 to 292 of human p53 protein and at least one intragenic suppressor mutation. The intragenic suppressor mutation is capable of suppressing a mutation of human p53 that occurs in human cancers when the intragenic suppressor and the cancer mutations are present in a cis configuration. The set is selected from the group consisting of: T81S; A83V; P87R; Q100L; Q100R; Q104P; F113L; L114V; T118M; V122I; C124S; K139R; Q144L; W146R; Q165L; V172I; H178Y; S183T; A189V; F212L; E224G; S227P; S227T; D228N; D228A; D228E; C229W; T230S; T231I; I232V; H233R; H233Y; Y234F; N235K; N235S; Y236N; N239M; N239W; N239L; N239F; N239R; N239H; S240Q; S240T; S240R; D281G; E285G; E285K; E294G; G325R; E343V; E346G; D352G; and combinations thereof.  
         [0025]     In a further embodiment of the invention a human p53 polypeptide is provided. The polypeptide has a portion of human p53 protein. The portion comprises residues 94 to 292 of human p53 protein and a set of at least one intragenic suppressor mutation. The set is capable of suppressing a mutation of human p53 that occurs in human cancers when the mutation is present in a cis configuration to the cancer mutation. The set is selected from the group consisting of: F113L; L114V+T123P+V172I+A189V; T123P+A189V; S227P+N239Y; T118M+H168R; V122I+C124S+H168R; T123A+H168R; K139R+H168R+N239Y; H168R+T231I; T123A+S240R; H178Y+S240R; D281G+E285G+G325R+E343V; E285K+D352G; E285K+E294G+E346G; T81S+A83V+S240R; P87R+Q100L+Q104P+Q144L+S240R; Q100R+W146R+S240R; Q165L+F212L+S240R; S183T+S240R; E224G+S240R; S240R; D228A+N235K+N239M; D228E+N235K+Y236N+N239L; I232V+H233R+Y234F+N235K+N239L; N235K+S240T; Y234F+N239L; N235K+N239R; H233Y+N239Y; N239F; N239Y+S240Q; H233Y+N235K+N239Y; N235K+N239Y; N235S+N239Y+S240N; D228N+N239Y; N235K+N239W; D228E+N239Y; N235K+S240N; N239W; T230S+N239Y; S227T+N235K+N239Y; H233R+N235S+N239R+S240R; N239R; N239R+S240R; C229W+N239R+S240R; N239L+S240R; N239F+S240R; I232V+N239H+S240R; D228E+C229W+N235K+N239Y+S240R; N239Y+S240R; and combinations thereof.  
         [0026]     These and other embodiments of the invention, which will be apparent to those of skill in the art, provide the art with reagents to suppress mutant p53 for the treatment of cancer. P53 suppressors and vectors for gene therapy treatment of cancers that contain p53 mutations are also provided. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0027]      FIGS. 1A and 1B . Structure of the p53 core DNA-binding domain and pattern of missense mutations within the core domain.  FIG. 1A  shows the structure of the p53 core domain. A β sandwich serves as the scaffold for two large loops (termed L2 and L3) and a loop-sheet-helix motif. The loops and the loop-sheet-helix motif form the DNA binding surface of p53. The L3 loop makes DNA contacts in the minor groove, while the H2 α helix and the L1 loop of the loop-sheet-helix motif make contacts in the major groove. The L2 and L3 loops provide stability to the DNA-binding surface through interactions with a Zn atom (Cho et al., 1994.)  FIG. 1B  is a map of the pattern of missense mutations within the p53 core domain. The vast majority of tumor-derived p53 missense mutations map to the p53 core domain as shown by the mutation histogram superimposed on the schematic p53 protein. Six “hot spot” codons are preferentially mutated due to exogenous carcinogens and endogenous biological processes. Mutations in the N-terminal transactivation and the C-terminal tetramerization domain are exceedingly rare. The white box in the very C-terminus indicates the location of the autoregulatory domain.  
         [0028]      FIGS. 2A, 2B ,  2 C, and  2 D show the design and characterization of a new p53 open reading frame.  FIG. 2A  is a comparison of the p53 open reading frame before and after cloning to introduce multiple restriction sites by silent mutagenesis. Before cloning of the new open reading frame, suppressor mutations with the most frequent p53 cancer mutations required a significant amount of subcloning.  FIG. 2B  shows that the new pRB500 expression plasmid (designer-p53 gene) and pRB16 (native p53 gene) have the same phenotype, Ura + Foa S , in a yeast strain with the reporter gene 1cUAS53::URA3.  FIG. 2C  shows that pRB500 and pRB16 have equal growth in SC-Ura media. A control strain with the reporter gene alone does not grow, while a strain with the URA3 gene shows superior growth.  FIG. 2D  shows that the new p53 expression plasmid pRB500 leads to similar p53 protein levels in yeast, as compared to the previously used pRB16.  
         [0029]      FIG. 3  shows the p53 cancer mutation to be analyzed. The p53 cancer mutations were chosen by their relative frequency in human cancer. All eight cancer mutations that will be used for the search of suppressor mutations are located at “hot spot” codons, codons that have a particularly high frequency of mutations. The first set and the second set of mutations (chosen for the subcloning analysis) comprise approximately 37% of all human cancers with p53 mutations. This is estimated to correspond to 123,000 cancer patients per year in the United States and 890,000 cancer patients worldwide (Harris, 1996a, 1996b.) 
     
    
       [heading-0030]     Table Legends  
         [0031]     Table 1. Intragenic suppressor mutations obtained with PCR mutagenesis. All clones shown in the table are derived from independent PCR-mediated mutational events. For Arg249Ser and Arg273Cys, all isolated clones are shown. For Gly245Ser and Arg273His, only a small subset of positive clones that were sequenced is shown. Mutations with a “*” have been previously isolated, alone or in combination, with cancer mutations Gly245Ser or Arg249Ser ( EMBO J.  17, 1847). Mutations in bold led to our hypothesis of a “suppressor motif” around amino acids 239 and 240 of the p53 protein.  
         [0032]     Table 2. Intragenic suppressor mutations obtained with oligonucleotide-based mutagenesis. “His + ” indicates the total amount of tansformants for a p53 cancer mutation library. “Ura + ” indicates the amount of Ura +  colonies (including false-positives), obtained from the total amount of His +  transformants. The columns with numbers correspond to codon numbers of the p53 ORF for which mutations were identified upon sequencing of Ura +  clones (the mutagenized codons were 225 to 241). The following p53 cancer mutants (15 of 31) were not rescued (His +  colonies×10 5 /Ura colonies in parentheses): C135Y (7.3.0), P151S (4.8/0), R175H (2.2/0), C176F (3.4/0), H179R (5.7/0), H179Y (5.3/0), G245D (1.4/0), R248Q (1.2/10), R248L (1.9/0), R248W (1.8/1), R249S (2.0/1), P278L (1.1/0), R280T (6.5/0), R282W (3.3/9) and E285K (not done). Our analysis targeted the 35 most frequent p53 cancers mutations based on three international databases ( Nucleic Acids Res.  26, 198, 200 &amp; 205). Y234C, M237I, C238Y and S241F had to be excluded, since they reside within the region that was mutagenized. An update of one of the databases ( Hum. Mutat.  14, 1) reported several additional cancer mutations that were as frequent as our chosen top 35. There were reported too late for our analysis (R158H, C176Y, I195T, G214R, G245V, G266R, G266E, P278S and R280K).  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0033]     The inventor has discovered intragenic suppressor motifs in the p53 protein. Surprisingly the motifs appear to restore function to many of the most common p53 cancer mutations. Gene therapy vectors containing nucleic acids encoding the intragenic suppressor motif can be used to deliver p53 containing a suppressor motif to cells containing mutant p53. The p53 molecules containing a suppressor motif can be used for protein crystallography to determine a protein stabilization mechanism. Such studies can be used to model and design small molecules that have the same function. Such small molecules can be used as cancer therapeutics.  
         [0034]     A nucleic acid molecule encoding human p53 containing at least one intragenic suppressor mutation may also contain a mutation found in human cancer. The intragenic suppressor mutation is capable of suppressing a human cancer mutation when both mutations are present in a cis configuration. The nucleic acid may contain a single mutation selected from the following mutations: T81S; A83V; P87R; Q100L; Q100R; Q104P; F113L; L114V; T118M; V122I; C124S; K139R; Q144L; W146R; Q165L; V172I; H178Y; S183T; A189V; F212L; E224G; S227P; S227T; D228N; D228A; D228E; C229W; I230S; T231I; I232V; H233R; H233Y; Y234F; N235K; N235S; Y236N; N239M; N239W; N239L; N239F; N239R; N239H; S240Q; S240T; S240R; D281G; E285G; E285K; E294G; G325R; E343V; E346G; and D352G. The isolated nucleic acid can also contain any combination of these mutations. The combination of intragenic suppressor mutations can include at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 of the mutations. Other mutations, whether cancer or suppressor may also be present on the nucleic acid.  
         [0035]     The nucleic acid molecule can alternatively contain a set of at least one intragenic suppressor mutation. The set of at least one intragenic suppressor mutation can be any one of the following sets of mutations: F113L; L114V+T123P+V172I+A189V; T123P+A189V; S227P+N239Y; T118M+H168R; V122I+C124S+H168R; T123A+H168R; K139R+H168R+N239Y; H168R+T231I; T123A+S240R; H178Y+S240R; D281 G+E285G+G325R+E343V; E285K+D352G; E285K+E294G+E346G; T81S+A83V+S240R; P87R+Q100L+Q104P+Q144L+S240R; Q100R+W146R+S240R; Q165L+F212L+S240R; S183T+S240R; E224G+S240R; S240R; D228A+N235K+N239M; D228E+N235K+Y236N+N239L; I232V+H233R+Y234F+N235K+N239L; N235K+S240T; Y234F+N239L; N235K+N239R; H233Y+N239Y; N239F; N239Y+S240Q; H233Y+N235K+N239Y; N235K+N239Y; N235S+N239Y+S240N; D228N+N239Y; N235K+N239W; D228E+N239Y; N235K+S240N; N239W; T230S+N239Y; S227T+N235K+N239Y; H233R+N235S+N239R+S240R; N239R; N239R+S240R; C229W+N239R+S240R; N239L+S240R; N239F+S240R; I232V+N239H+S240R; D228E+C229W+N235K+N239Y+S240R; N239Y+S240R. The isolated nucleic acid can also contain any combination of these sets of mutations. The combination of sets of mutations can include at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45, of the sets of intragenic suppressor mutations.  
         [0036]     The nucleic acids of the invention can be deoxyribonucleic acids (DNA) or ribonucleic acid (RNA) molecules such as mRNA. The nucleic acids can be linear nucleic acids or if the nucleic acids are DNA, they can be cloned into any suitable vector. Suitable vectors include plasmids, artificial chromosomes, or viral genomes. Plasmids are well known in the art and include plasmids that are suitable for introduction of the p53 gene into bacterial, yeast, mammalian, insect, or other eukaryotic cells or organisms. The plasmids may be available through a commercial vendor, or may be noncommercial plasmids, or derivatives thereof. Artificial chromosomes are preferably the artificial chromosomes of humans, yeast, or bacteria. Viral vectors are also well known in the art. Preferably the viral genome is the genome of an adeno-associated virus, adenovirus, herpes virus, retrovirus, or vaccinia virus. Viral vectors such as Baculovirus may also be used for subsequent use in insect cells.  
         [0037]     p53-encoding nucleic acids can be introduced into a vector by any molecular biology technique known for this purpose. Several nonlimiting examples of such techniques include restriction enzyme digestion of the p53 nucleic acids and direct ligation into a vector, or PCR amplification of the p53 nucleic acids and subsequent cloning by restriction enzyme digestion and ligation into a vector. Other techniques for cloning the p53 nucleic acids into a vector can be found in Sambrook, J., Fritsch, E. and Maniatis, T.,  Molecular Cloning: A Laboratory Manual  Second Edition. Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and Ausubel, F. M. et al.,  Current Protocols in Molecular Biology . Wiley, Interscience New York (1987). Methods of mutagenesis of the p53 gene can also be found in these references.  
         [0038]     It is also contemplated that the nucleic acid encoding p53 with an intragenic suppressor mutation can further comprise regulatory sequences that enhance the expression of the p53 gene containing the intragenic suppressor mutation. Promoters and enhancers are well known to those of skill in the art. Several nonlimiting examples include constitutive promoters such as the strong promoters of cytomegalovirus, SV40, or Rous sarcoma virus. Promoters can also be inducible promoters that are induced by drugs like tetracycline, or tissue specific promoters. Tissue specific promoters include the albumin promoter for expression in the liver, the myosin light chain 1 promoter for expression in muscle and endothelial cells, the surfactant protein A or keratin 18 for expression in lung, and the prostate specific antigen (PSA) promoter, the probasin (PB) promoter, or the prostate specific membrane antigen promoter for expression in prostate. Tumor specific promoters can also be used. Tumor specific promoters include the tyrosine kinase promoter for B16 melanomas, the DF3/MUC1 promoters for certain breast cancers, and the α fetoprotein promoter of hepatomas. Enhancers may be used from viruses such as Rous sarcoma virus, hepatitis B virus, or simian virus-40 can be used. Enhancers from the cyclic AMP response element, serum response element, nuclear factor kappa b element, activator protein 1, or serum response factor may be used as well. Any enhancers known in the art can be used.  
         [0039]     The nucleic acids encoding human p53 may further comprise a selectable marker gene. The selectable marker gene allows easy detection of the transfer of the p53 nucleic acids into a suitable host cell. Selectable marker genes may be genes that confer resistance to a toxic agent such as an antibiotic. Antibiotic resistance genes include those for ampicillin, tetracycline, puromycin, neomycin, and hygromycin. The selectable marker gene may confer resistance to a toxic agent; such genes include the adenine deaminase, aminoglycoside phosphotransferase, dehydrofolate reductase or xanthine-guanine phosphoribosyltransferase gene. The selectable marker gene may alternatively be a reporter gene whose expression is monitored readily by assay. Several nonlimiting examples of such reporter genes are chloramphenicol transacetylase, firefly luciferase, beta galactosidase, secreted alkaline phosphatase, and beta glucuronidase. The marker gene can also be a gene that allows growth of a cell on medium lacking an amino acid. An example of a selectable marker gene of yeast is the URA3 gene. Counterselectable genes can also be used with yeast such as LYS2, LYS5, CAN1, MET2, MET15, and GAL1. Other such marker genes are known in the art.  
         [0040]     The nucleic acid molecule may optionally encode at least one mutation of human p53 found in a cancer. The isolated nucleic acid may also encode at least two, at least three, at least five, or at least ten mutations of human p53 found in a cancer. Human cancers containing p53 mutations include tumors of the digestive organs, respiratory system, breast, female genital organs, head and neck, hematopoeitic system, skin, brain, bladder, male genital organs, soft tissues, bone and others. Mutations of human p53 found in cancer include, but are not limited to: R175H, G245S, R248Q, R248W, R249S, R273C, R273H, R282W, C135Y, C141Y, P151S, V157F, R158L, Y163C, V173L, V173M, C176F, H179R, H179Y, H179Q, Y205C, Y220C, Y234C, M237I, C238Y, S241F, G245D, G245C, R248L, R249M, V272M, R273L, P278L, R280T, E285K, E286K, R158H, C176Y, I195T, G214R, G245V, G266R, G266E, P278S, and R280K. Preferably, the mutation found in a cancer is selected from the following: G245S; R249S; R273C; R273H; C141Y, V157F, R158L, Y163C, V173L, V173M, Y205C, Y220C, G245C, R249M, V272M, R273L, and E286K.  
         [0041]     The nucleic acids of the invention can also be isolated or in a cell. The cells can be of any type including mammalian cells, insect cells,  Drosophila  cells, yeast cells or bacterial cells. If the cells are mammalian cells, they can be the cells of any species including humans, mice, monkeys, pigs, rats, cows, horses, cats, or dogs. The mammalian cells can further be manipulated to knock out one or both endogenous copies of the cell&#39;s p53 genes encoded in its cellular DNA, thus allowing study of the mutant human p53 gene alone in the cells.  
         [0042]     The mammalian cells can be in the body of a mammal or may be in in vitro culture. If the cells are in culture, the cells may be primary cells or may be a stable cell line. The cells may also contain a wild type p53 gene or may have a p53 gene encoding a p53 mutation that has been identified as being associated with a human cancer, a p53 mutation that has not yet been associated with a human cancer, or a p53 mutation that is not associated with a human cancer. The cells may also be tumor cells that may or may not contain a mutant p53 gene. Such cells can be used for producing a suppressor protein for drug screening or crystallographic purposes. The p53 molecules containing an intragenic suppressor mutation or set of intragenic suppressor mutations may also suppress in trans a p53 mutation found in human cancers.  
         [0043]     The human p53 nucleic acids may be introduced into the cells by any means known in the art. The nucleic acids may be inserted into the cells by direct transfer, by microinjecting the nucleic acids into the cells. The nucleic acids may also be complexed in a lipid preparation such as liposomes, or coacervated with a polymeric cation. The nucleic acids may alternatively be transferred into cells by electroporation, using DEAE dextran or calcium phosphate. The human p53 nucleic acids may further be transferred into cells using viruses with suitable characteristics for entry into the cells. The transfer of the p53 nucleic acids into the cells may achieve stable or transient transfection.  
         [0044]     A human p53 protein according to the invention can be isolated and purified. An isolated and purified human p53 protein is a protein that is mostly free of contaminating proteins. The protein is preferably 60%, more preferably 70 or 75%, even more preferably 80% or 85%, or most preferably 90%, 95%, 96%, 97%, 98%, or 99% pure with respect to other proteins. It is understood that buffers, salts, detergents, or other molecules that promote the stability or enhance the activity of the protein may be present with the isolated and purified human p53.  
         [0045]     The protein of the invention can be introduced into the cells by any means known in the art. The protein can be expressed after introduction of DNA encoding the protein or by direct introduction of the protein using techniques such as microinjection. The cells may also take up the protein passively, or the cells may take up the protein after fusion to another protein that expedites cellular uptake. Such proteins include the tat protein of human immunodeficiency virus, or a portion of tat that contains amino acid residues 49-57. These proteins also include the amino-terminal portion of the Anthrax lethal factor, preferably amino acid residues 1-255 of the lethal factor or amino acid residues 1-255 with short tracts of lysines, arginines, or histidines fused to its N-terminus. Also the protein may be fused to the amino-terminal portion of the Diphtheria toxin enzymatic A chain from residues 1-193, or to this portion of the enzymatic A chain with short tracts of lysines, arginines, or histidines fused to its N-terminus.  
         [0046]     The protein of the invention may also be a portion of the human p53 protein, i.e., less than the full-length protein found in humans having 393 amino acid residues. The portion of the p53 protein may preferably contain at least residues 81 to 292, 81 to 300, 81 to 312, 81 to 352, 94-292, 94 to 300, 94 o 312, or 94 to 352 of human p53.  
         [0047]     While the invention has been described with respect to specific examples including presently preferred modes of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the above described systems and techniques that fall within the spirit and scope of the invention as set forth in the appended claims.  
       EXAMPLES  
     Example 1  
       [0048]     Identification and evaluation of intragenic suppressor mutations for the most common p53 cancer mutations. Our previous study clearly established that various mechanisms for stabilizing the p53 core domain exist; and it is very likely that additional ones are waiting to be discovered (Brachmann et al., 1998). In light of the discovered p53 suppressor mutations and the need for a comprehensive analysis for the most common p53 cancer mutations, we have designed a much-improved strategy that will require more initial effort, but dramatically streamline future analyses. The design is based on several shortcomings that we encountered and includes the synthesis of a new p53 ORF with multiple restriction sites, as well as specific codon choices for p53 cancer mutations to allow for easy secondary screens.  
         [0049]     Once we had isolated the suppressor mutations in our pilot study, we were very interested in establishing whether these mutations would be able to suppress other cancer mutations. This was fairly easily achieved for mutations such as T123A and T123P yet very cumbersome for N239Y, S240N and N268D due to the scarcity of naturally present restriction sites. We therefore synthesized an entirely new p53 ORF that includes a multitude of unique restriction sites or sites with one additional site in the vector. The introduction of restriction sites by silent mutagenesis was performed using the program WebCutter 2.0 (http://www.firstmarket.com/cutter/cut2.html) and the result is shown in  FIG. 2A .  
         [0050]     The new ORF was assembled from four fragments that were 379, 249, 257 and 325 base pairs long. Between 3 and 8 clones were sequenced for each of the p53 ORF fragments. This identified clones without mutations for 2 of the 4 fragments. The third fragment could be constructed by combining the areas of two clones without mutations. The fourth fragment was constructed by repairing a single mutation with oligonucleotides. After assembling the entire new p53 ORF with the ADH1-promoter and CYC1-terminator in pRS413 (CEN/HIS3), we compared the new pRB500 with the previously used p53 expression plasmid pRB16 ( FIG. 2 ).  
         [0051]     Both p53 expression plasmids showed the same phenotype in the presence of the p53-responsive URA3 reporter gene 1cUAS53::URA3 ( FIG. 2B ). Four independent transformants were Ura + Foa S , while controls lacking either a p53 expression plasmid or the reporter gene had the opposite phenotype, Ura − Foa R . All strains grew on SC-His plates, indicating the presence of the plasmid with the marker gene HIS3.  
         [0052]     Comparison of the growth rates in SC-Ura media confirmed the results of the plating assays: both plasmids lead to the expression of similar amounts of p53 protein ( FIG. 2C ). Western Blot analysis using a monoclonal anti-p53 antibody showed that pRB500 indeed leads to p53 protein levels that are similar to and maybe slightly higher than those with pRB16 ( FIG. 2D ).  
         [0053]     This new p53 ORF is also optimized as much as possible for the preferential codon usage of  E. coli , yeast and mammalian cells (Zhang et al., 1991; Wada et al., 1992). To make diagnostic restriction digestions easier to interpret, we also destroyed a variety of restriction sites.  
       Example 2  
       [0054]     Identification of suppressor mutations for the eight most common p53 cancer mutations. Our pilot study explored the feasibility of our genetic strategy in yeast and, thus, did not attempt a systematic search for suppressor mutations up- and downstream of a representative set of p53 cancer mutations. A comprehensive analysis is needed to better understand the structural dynamics of the p53 core domain and to potentially find suppressor mutations that have a universal effect. We chose to study the most frequent p53 cancer mutations. Based on several international databases for p53 cancer mutations (Beroud and Soussi, 1998; Cariello et al., 1998; Hainaut et al., 1998), we selected a total of eight mutations ( FIG. 3 ). Each of these cancer mutations represents between 1.6% and 4.2% of all human cancers with p53 mutations, totaling 22% (estimated to represent 73,000 cancer patients per year in the United States and 530,000 cancer patients worldwide), (Harris, 1996a, 1996b). Besides their high frequency, these eight cancer mutations also well represent the most important structural motifs of the p53 core domain (L2 loop: codon 175, L3 loop: codons 245, 248 and 249, loop-sheet-helix motif: codons 273 and 282).  
         [0055]     We then performed a comprehensive search for intragenic suppressor mutations up- and downstream of the eight most common cancer mutations, R175H, G245S, R248Q, R248W, R249S, R273C, R973H and R282W, a distinct subset that accounts for 28% of all reported p53 missense mutations (Beroud, 1998; Cariello, 1998; Hainaut, 2000; Hernandez-Boussard, 1999). Briefly, we obtained PCR products under mutagenic conditions covering the core domain regions up- and downstream of a cancer mutation. We then transformed the PCR products with gapped expression plasmids for the cancer mutations into the yeast p53 reporter strain Rby377. Gap repair by homologous recombination in yeast reconstituted the expression plasmids and led to the isolation of suppressor and cancer mutation combinations that resulted in transcriptionally active p53 in yeast (Brachmann, 1996; Brachmann, 1998; Vidal, 1996). We were unable to identify suppressor mutations for R175H, R248Q, R248W and R282W, but isolated multiple suppressor mutation combinations for R245S, R249S, R273C and R273H (Table 1). Strikingly, half of all suppressor mutation combinations contained previously and newly isolated mutations in codons 239 and 240.  
                         TABLE 1                           Intragenic suppressor mutations obtained with PCR mutagenesis            Cancer Mutation   Suppressor Mutations               G245S   F113L 1             L114V + T123P* + V172I + A189V 2             T123P* + A189V 3             S227P + N239Y* 4             N239Y* 5         R249S   T118M + H168R* 6             V122I + C124S + H168R* 7             T123A* + H168R* 8             K139R + H168R* + N239Y* 9             H168R* + T231I 10         R273C   T123A* + S240R 11             H178Y + S240R 12             D281G + E2850 + G325R + E343V 13             E285K + D352G 14             E285K + E294G + E346G 15         R273H   T81S + A83V + S240R 16             P87R + Q100L + Q104P + Q144L + S240R 17             Q100R + W146R + S240R 18             Q165L + F212L + S240R 19             S183T + S240R 20             E224G + S240R 21             S240R 22                    
 
       Example 3  
       [0056]     Prediction of suppressor mechanisms based on the core domain crystal structure of wild-type p53. The crystal structure of the wild-type p53 core domain (Cho et al., 1994) has improved our understanding of p53 immensely (Friend, 1994; Prives, 1994; Vogelstein and Kinzler, 1994). Based on the wild-type p53 crystal structure, likely mechanisms of stabilization for the suppressor mutations identified so far is predicted (Brachmann et al., 1998). Our newly identified suppressor mutations will be subject to the same analysis.  
       Example 4  
       [0057]     Evaluation of all identified suppressor mutations with a total of 35 p53 cancer mutations in yeast. Besides the first set of eight p53 mutations, we selected 27 further mutations for this final evaluation ( FIG. 3 ). Each cancer mutation in the second set accounts for 0.04 to 1.1% of reported p53 mutations, totaling 15% of all human cancers with p53 mutations (estimated to represent 50,000 cancer patients per year in the United States and 360,000 cancer patients worldwide), (Harris, 1996a, 1996b). This second set reflects many different mechanisms of destabilizing the p53 core domain since the mutations locate to different structural motifs (β sandwich: 8; loop-sheet-helix motif: 7; L2 loop: 5; L3 loop: 7).  
         [0058]     Based on our results identifying suppressor mutations for the eight most common p53 cancer mutations, (Example 2), we hypothesized that the area of the p53 core domain around residues 239 and 240 represents a “suppressor motif” that may restore function to other p53 cancer mutants as well. Since PCR mutagenesis yields only a subset of all possible codon changes, we decided to focus on this area using oligonucleotide-mediated mutagenesis. We constructed a library of annealed oligonucleotides that encoded for all possible 19 amino acid changes in codons 239 and 240. In addition, the other codons were synthesized under mutagenic conditions, resulting in approximately one in 100 nucleotide misincorporations. The annealed oligonucleotides represented codons 225 to 241 and were cloned into gapped expression plasmids for 30 of the most common p53 cancer mutations. These expression plasmid libraries were then transformed into the yeast p53 reporter strain and Ura +  colonies selected. More than half (16 of 30) of the p53 cancer mutants tested were rescued ( FIG. 1   b  and Table 2). A total of 39 independent suppressor mutation combinations were isolated. Besides the expected amino acid changes in codons 239 and 240, mutations in codon 235, mainly to Lysine, were repeatedly isolated in combination with codon 239 or 240 changes or both (Table 2).  
                                                                                                           TABLE 2                           Intragenic suppressor mutations obtained with oligonucleotide-based       mutagenesis            Cancer   His +         228   232   233   234   235   239   240   Other       Mutation   (×10 5 )   Ura +     D   I   H   Y   N   N   S   mutations                    C141Y   6.5   25   A               K   M         23                      E               K   L       Y236N 24                         V   R   F   K   L         25                                     K       T     26         V157F   7.9   215               F       L         27                                     K   R         28                                         Y         29         R158L   3.7   437           Y           Y         30                                         F         31                                         Y         32                                         Y   O     33         Y163C   3.3   4           Y       K   Y         34                                     K   Y         35                                     S   Y   N     36         V173L   3.5   2,560   N                   Y         37                                         Y         38         V173M   3.5   2                   K   Y         39         Y205C   3.6   650                   K   W         40                     Q                   Y         41         Y220C   1.3   5,890                       Y         42                                     K       N     43         G245C   2.3   339                       W         44                                         Y         45         G245S   1.2   3,300                       F         46                                         Y       T230S 47         R249M   1.5   109                   K   Y       S227T 48                                         Y         49         V272M   2.4   3,560                       W         50         R273C   2.3   290           R       S   R   R     51                                         R         52                                         R   R     53                                         R   R   C229W 54                                         L   R     55                                         F   R     56         R273H   3.1   2,100       V               H   R     57                                             R     58         R273L   1.3   19   Q               K   Y   R   C229W 59                                         Y   R     60         E286K   5.7   1                   K   W         61                     
 
         [0059]     We indeed confirmed codons 239 and 240 as an important general suppressor motif whose effect appears to be further enhanced by codon 235. With changes in these three amino acids, we were able to rescue 16 of 31 p53 cancer mutants tested. The rescued p53 mutations are located within the β-sandwich (codons 141, 157, 158, 163, 205 and 220), the L2 loop (codon 173), the L3 loop (codons 245 and 249) and the loop-sheet-helix motif (codons 272, 273 and 286) (Cho, 1994), supporting the idea of a suppressor motif with a general rescue mechanism. This suggests that the 235-239-240 suppressor motif will restore function to many more of the reported over 900 different p53 cancer mutants. The collection of p53 cancer mutants that we successfully rescued in this study represents 13.7% of all entries in an international p53 mutation database (Hernandez-Boussard, 1999), estimated to amount to 330,000 patients every year worldwide (Harris, 1996a).  
         [0060]     Different suppressor mutation combinations were isolated. Yet, it is very likely that many combinations will result in a very similar rescue mechanism, considering that 87% of codon 235 changes are lysine, 61% of 239 changes either tyrosine or arginine and 69% of 240 changes arginine. Based on previous predictions and studies, our general suppressor motif may provide additional DNA contacts or may increase the thermodynamic stability of the core domain (Brachmann, 1998; Nikolova, 2000). Crystallographic studies are under way to determine the exact suppressor mechanism. In this context, some of the less common suppressor mutation combinations may be particularly helpful (see, for example, Table 2, #s 23, 31 and 57), since they may result in similar conformational changes as the more frequent ones, but by other means.  
       Example 5  
       [0061]     Mammalian assays to demonstrate biological activity of p53 double mutants. Our previous study clearly established a very tight correlation between yeast results and subsequent mammalian assays (Brachmann et al., 1998). We found some minor inconsistencies when we used mammalian reporter gene assays; however, the lacZ transfection viability assay showed 100% correlation between the yeast results and the mammalian assay. We will therefore perform mammalian assays only for a subset of p53 double mutants (one suppressor and one cancer mutation). The mutation combinations will be chosen based on the above criteria (interesting and/or universal suppressor mechanisms, most frequently encountered cancer mutations) and subcloned into the pCMVneo-based p53 expression plasmid, pC53-SN3 (Baker et al., 1990).  
       Example 6  
       [0062]     Determination of the core domain crystal structures for selected p53 proteins containing a suppressor, a cancer or both mutation(s). In brief, residues 94-312 of the core domains will be produced in  E. coli  BL21 (D3) using the pET3d expression vector (Novagen). The cell lysate will be loaded onto a Mono S cation-exchange column (Pharmacia) and eluted by a NaCl gradient. The p53-containing Mono S fraction will be precipitated by 80% saturated ammonium sulfate and purified further by gel-filtration chromatography on a Superdex 75 gel-filtration column (Pharmacia), yielding the protein at &gt;98% purity (Pavletich et al., 1993; Cho et al., 1994). Some p53 single and double mutants may have different purification properties for which we will try modified purification protocols for them. The purified materials will then be grown into crystals and their structure determined.  
         [heading-0063]     Materials and Methods  
         [0064]     Transcriptional activity of wild-type p53 in yeast. This assay scores for the transcriptional activity of wild-type p53 and uses an artificial reporter gene, 1cUAS53::URA3, with a synthetic consensus p53 DNA-binding site upstream of URA3 (Brachmann et al., 1996; Vidal et al., 1996). Human p53 is expressed from a yeast CEN expression plasmid under the control of the constitutive promoter ADH1. This p53 yeast assay is unique in that it not only allows selection for, but also against functional p53 (Brachmann and Boeke, 1997). Therefore, it can quickly classify any p53 protein for its activity: p53 proteins with wild-type p53 activity induce URA3 expression which enables the yeast reporter strain to survive on plates without Uracil (Ura + ), but also sensitizes the strain to 5-fluoro-orotic acid (5-Foa) resulting in the second phenotype, Foa sensitivity (Foa S ). P53 cancer mutants that have lost function have the opposite phenotype of wild-type p53, Ura − Foa R  (Brachmann et al., 1996). p53 proteins with partial loss of wild-type p53 function can be easily detected as well since they have the intermediate Ura + Foa R  phenotype. This reflects sufficient URA3 expression for survival on SC-Ura plates, but insufficient expression to be sensitive to 5-Foa.  
         [0065]     Engineered p53 open reading frame. A new ORF for wild-type p53 with multiple silent restriction sites and optimized for codon usage in bacteria, yeast and mammalian cells (Wada, 199; Zhang, 1991) was designed with WebCutter 2.0 (http://www.firstmarket.com/cutter/cut2.html) and constructed using the “KAPPA” method (Holowachuk, 1995). When compared with the natural p53 yeast expression plasmid pRB16, the engineered p53 ORF in the same yeast plasmid resulted in two-fold more p53 protein and identical yeast phenotypes (Brachmann, 1996; Brachmann, 1998; Vidal, 1996). Annealed oligonucleotides encoding for the most common p53 cancer mutations (see Table 2) were cloned into the engineered p53 ORF.  
         [0066]     PCR- and oligonucleotide-mediated mutagenesis strategy to identify intragenic suppressor mutations for p53 cancer mutations. PCR-mediated mutagenesis was performed as previously described (Brachmann, 1998), except that engineered p53 ORFs for the p53 cancer mutants, mutagenic PCR conditions (Lin-Goerke, 1997; Svetlov, 1998) and Rby377, a diploid yeast strain with two copies of the p53-dependent URA3 reporter gene, were used. A library of annealed oligonucleotides that equally represented all possible amino acid changes in codons 239 and 240 and had approximately one in 100 additional nucleotides mutated were cloned into PflMI-NsiI gapped yeast expression plasmids for p53 cancer mutants, transformed into Rby377 and analyzed as described (Brachmann, 1998).  
         [0067]     Yeast and mammalian assays for p53. Yeast assays for p53 and mammalian reporter gene assays were performed as described (Brachmann, 1996; Brachmann, 1998; Vidal, 1996).  
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