Suppressor mutations for common P53 cancer mutations

Abstract

Intragenic suppressor mutations of common p53 mutations are able to function in cis and/or trans. These mutations are useful for identifying small molecule drugs which function in a similar fashion. In addition, the mutations themselves may be useful therapeutically, especially if they function in trans. Methods for rapidly obtaining this type of mutant employ a yeast selection system. Cells having both the negative mutation and intragenic suppressor are useful for studying the interactions of the two, in particular in determining the structure of the homotetramers and heterotetramers.

1 . A nucleic acid encoding human p53 which carries: 
 at least one intragenic suppressor mutation, wherein said intragenic suppressor mutation suppresses a dominant-negative mutation of p53; and    at least one dominant negative mutation of p53.    
     
     
         2 . The nucleic acid encoding human p53 of  claim 1  wherein the intragenic suppressor mutation is T123P.  
     
     
         3 . The nucleic acid encoding human p53 of  claim 1  wherein the intragenic suppressor mutation is N268D.  
     
     
         4 . The nucleic acid encoding human p53 of  claim 1  wherein the intragenic suppressor mutation is N239Y.  
     
     
         5 . The nucleic acid encoding human p53 of  claim 1  wherein the intragenic suppressor mutation is S240N.  
     
     
         6 . The nucleic acid encoding human p53 of  claim 1  wherein the intragenic suppressor mutation is T123A.  
     
     
         7 . The nucleic acid encoding human p53 of  claim 1  wherein the at least one intragenic suppressor mutation comprises a T123A and a H168R mutation.  
     
     
         8 . The nucleic acid encoding human p53 of  claim 1  wherein the intragenic suppressor mutation is not located in α-helix H2.  
     
     
         9 . The nucleic acid encoding human p53 of  claim 1  wherein the intragenic suppressor mutation is located in a loop selected from the group consisting of L1, L2, and L3.  
     
     
         10 . The nucleic acid encoding human p53 of  claim 1  wherein the intragenic suppressor mutation is located in α-helix H1.  
     
     
         11 . The nucleic acid encoding human p53 of  claim 1  wherein the intragenic suppressor mutation is located in a β-strand domain.  
     
     
         12 . The nucleic acid encoding human p53 of  claim 1  which comprises at least two intragenic suppressor mutations.  
     
     
         13 . A nucleic acid encoding human p53 which carries: 
 at least one intragenic suppressor mutation, wherein said intragenic suppressor mutation suppresses a dominant-negative mutation of p53, wherein the at least one intragenic suppressor mutation is selected from the group consisting of: T123P, N268D, N239Y, S240N, and T123A+H168R.    
     
     
         14 . A cell comprising: 
 a first nucleic acid which encodes human p53 and which comprises a dominant-negative mutation; and    a second nucleic acid encoding human p53 which carries at least one intragenic suppressor mutation, wherein said mutation suppresses a dominant-negative mutation of p53 in trans.    
     
     
         15 . The cell of  claim 14  which is eukaryotic.  
     
     
         16 . The cell of  claim 14  which is a yeast cell.  
     
     
         17 . The cell of  claim 14  which is a human cell.  
     
     
         18 . The cell of  claim 14  wherein the intragenic suppressor mutation suppresses a mutation selected from the group consisting of: V143A, R175H, G245D, G245S, R248W, R249S, R248Q, R273H, R273C, R282W, and R273P.  
     
     
         19 . A cell comprising: 
 a first nucleic acid which encodes human p53 and which comprises a mutation found in a tumor; and    a second nucleic acid encoding human p53 which carries at least one intragenic suppressor mutation, wherein said mutation suppresses a dominant-negative mutation of p53 in trans.    
     
     
         20 . The cell of  claim 19  which is eukaryotic.  
     
     
         21 . The cell of  claim 19  which is a yeast cell.  
     
     
         22 . The cell of  claim 19  which is a human cell  
     
     
         23 . The cell of  claim 19  wherein the intragenic suppressor mutation suppresses a mutation selected from the group consisting of: V143A, R175H, G245D, G245S, R248W, R249S, R248Q, R273H, R273C, R282W, and R273P.  
     
     
         24 . A cell which has been transfected with the nucleic acid encoding human p53 of  claim 1 .  
     
     
         25 . A method of treating cancer cells comprising the step of: 
 introducing into the cancer cells a nucleic acid encoding human p53 which carries at least one intragenic suppressor mutation, wherein said mutation suppresses a dominant-negative mutation of p53 in trans, whereby a neoplastic phenotype of the cancer cells is suppressed or apoptosis is induced.    
     
     
         26 . The method of  claim 25  wherein the cancer cells comprise a p53 gene which comprises a dominant-negative mutation.  
     
     
         27 . The method of  claim 25  wherein the cancer cells comprise a p53 gene which comprises a p53 mutation found in a tumor.  
     
     
         28 . A method for identifying mutations in a human p53 gene which suppress the phenotype of a dominant-negative p53 mutation, comprising the steps of: 
 introducing into a cell which carries a dominant-negative mutation in a p53-encoding DNA, a nucleic acid encoding p53 which has been mutagenized;    testing the cell to determine whether its phenotype is that of a cell carrying a wild-type p53 or a cell carrying a dominant-negative p53 mutation; wherein a cell whose phenotype is that of a wild-type p53 is a cell carrying a p53 suppressor mutation on the mutagenized nucleic acid encoding p53.    
     
     
         29 . The method of  claim 28  wherein the cell is a yeast cell.  
     
     
         30 . The cell of  claim 28  which is eukaryotic.  
     
     
         31 . The cell of  claim 28  which is a human cell  
     
     
         32 . The method of  claim 28  wherein the phenotype is determined by observing expression of a gene whose expression is p53-dependent.  
     
     
         33 . The method of  claim 32  wherein the p53-dependent gene is selected from the group consisting of p21 WAF1 , GADD45, IGF-BP3, and bax.  
     
     
         34 . The method of  claim 32  wherein the p53-dependent gene is a reporter gene under the control of a p53 responsive transcriptional element.  
     
     
         35 . The method of  claim 28  wherein the dominant-negative mutation is selected from the group consisting of: V143A, R175H, G245D, G245S, R248W, R249S, R248Q, R273H, R273C, R282W, and R273P.  
     
     
         36 . The method of  claim 28  wherein the nucleic acid encoding p53 was mutagenized using PCR-generated fragments to recombine with a gapped p53-encoding molecule.  
     
     
         37 . A method for identifying mutations in a human p53 gene which suppress the phenotype of dominant-negative p53 mutations, comprising the step of: 
 determining the phenotype of a cell which comprises a nucleic acid encoding p53 which carries a dominant-negative mutation and which has been mutagenized, if the phenotype of the cell is that of a cell carrying a wild-type p53 then the cell is a candidate cell for carrying a p53 suppressor mutation on the mutagenized p53 cDNA.    
     
     
         38 . The method of  claim 37  wherein the cell is a yeast cell.  
     
     
         39 . The cell of  claim 37  which is eukaryotic.  
     
     
         40 . The cell of  claim 37  which is a human cell  
     
     
         41 . The method of  claim 37  wherein the phenotype is determined by observing expression of a gene which is dependent on p53 for expression.  
     
     
         42 . The method of  claim 41  wherein the p53-dependent gene is selected from the group consisting of p21 WAF1 , GADD45, IGF-BP3, and bax.  
     
     
         43 . The method of  claim 41  wherein the p53-dependent gene is a reporter gene under the control of a p53 responsive transcription element.  
     
     
         44 . The method of  claim 37  wherein the dominant-negative mutation is selected from the group consisting of: V143A, R175H, G245D, G245S, R248W, R249S, R248Q, R273H, R273C, R282W, and R273P.  
     
     
         45 . The method of  claim 37  wherein the nucleic acid encoding p53 was mutagenized using PCR-generated fragments to recombine with a gapped p53-encoding molecule.