Tumor suppressor WAF1

Abstract

A human gene, WAF1, has been identified which is induced by wild-type but not mutant p53 in human brain tumor cells. The gene is located on chromosome 6p21.2 and directs the synthesis of an 18.1 kd protein. Introduction of WAF1 cDNA suppresses growth of human brain and colon tumor cells. The WAF1 gene and protein are useful inter alia for diagnosis and treatment of human tumors.

1 . An isolated and purified subchromosomal DNA molecule which encodes WAF1 protein as shown in SEQ ID NO: 2, said DNA molecule containing three exons of 168 bp, 450 bp and 1600 bp, wherein the sequence of said exons is shown in SEQ ID NO: 1.  
     
     
         2 . The DNA molecule of  claim 1  which is not more than 90 kb.  
     
     
         3 . The DNA molecule of  claim 1  which is a P1 clone.  
     
     
         4 . The DNA molecule of  claim 1  which contains at least two intervening sequences.  
     
     
         5 . The DNA molecule of  claim 1  which contains no intervening sequences.  
     
     
         6 . An isolated and purified WAF1 protein having a sequence as shown in SEQ ID NO: 2.  
     
     
         7 . An antibody which is specifically reactive with human WAF1 protein.  
     
     
         8 . A method of suppressing growth of tumor cells, comprising the step of: 
 administering to said cells a WAF1 protein having a sequence as shown in SEQ ID NO: 2.    
     
     
         9 . A method of suppressing growth of tumor cells, comprising the step of: 
 administering to said cells a DNA molecule which causes said cells to express WAF1, said DNA molecule having a sequence as shown in SEQ ID NO: 1.    
     
     
         10 . A method for screening potential therapeutic agents for the ability to suppress the growth of tumor cells by activating the expression of WAF1, comprising the steps of: 
 incubating a potential therapeutic agent with a cell which contains a WAF1 reporter construct, said reporter construct comprising a WAF1 transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable product;    measuring the production of the assayable product, a potential therapeutic agent which increases the production by the cell of the assayable product being an agent which will suppress the growth of tumor cells by activating the expression of WAF1.    
     
     
         11 . The method of  claim 10  wherein the WAF1 transcription regulatory region comprises about 2.4 kb upstream from WAF1 transcriptional start site.  
     
     
         12 . The method of  claim 10  wherein the WAF1 transcription regulatory region comprises the sequence of SEQ ID NO: 3.  
     
     
         13 . A method for diagnosing cancer, comprising the steps of: 
 testing a tissue to determine if the tissue expresses less WAF1 than normal tissue.    
     
     
         14 . The method of  claim 13  wherein the step of testing utilizes an antibody which is specifically reactive with WAF1 protein.  
     
     
         15 . The method of  claim 13  wherein the step of testing utilizes a nucleic acid probe which specifically hybridizes to a WAF1 mRNA, said probe having a sequence selected from SEQ ID NO: 1.  
     
     
         16 . A method for diagnosing cancer, comprising the steps of: 
 testing a tissue to determine if DNA in said tissue contains a mutant WAF1 gene.    
     
     
         17 . The method of  claim 16  wherein DNA of the tissue is compared to DNA of a normal tissue to determine whether the WAF1 gene is mutant.  
     
     
         18 . A WAF1 reporter construct, said reporter construct comprising a WAF1 transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable product.  
     
     
         19 . The reporter construct of  claim 18  wherein the WAF1 transcription regulatory region comprises about 2.4 kb upstream from WAF1 transcriptional start site.  
     
     
         20 . The reporter construct of  claim 18  wherein the WAF1 transcription regulatory region comprises the sequence of SEQ ID NO: 3.  
     
     
         21 . An antisense WAF1 construct comprising: 
 a. a transcriptional promoter;    b. a transcriptional terminator;    c. a DNA segment comprising one or more segments of the WAF1 gene, said gene segment located between said promoter and said terminator, said DNA segment being inverted with respect to said promoter and said terminator, whereby RNA produced by transcription of the DNA segment is complementary to a corresponding segment of WAF1 RNA produced by human cells.    
     
     
         22 . The antisense WAF1 construct of  claim 21  wherein said transcriptional promoter is inducible.  
     
     
         23 . A WAF1 antisense oligonucleotide comprising: at least ten nucleotides complementary to WAF1 mRNA.  
     
     
         24 . The WAF1 antisense oligonucleotide of  claim 23  which comprises at least about twenty nucleotides complementary to WAF1 mRNA.  
     
     
         25 . The WAF1 antisense oligonucleotide of  claim 23  which contains one or more modified nucleotide analogs.  
     
     
         26 . The WAF1 antisense oligonucleotide of  claim 23  which is a circular molecule.  
     
     
         27 . A method for promoting the proliferation of cells, comprising the step of: 
 administering a WAF1 antisense oligonucleotide comprising at least ten nucleotides complementary to WAF1 mRNA to said cells to inhibit the expression of WAF1.    
     
     
         28 . A method for promoting the proliferation of cells, comprising the step of: 
 administering a WAF1 triplex-forming oligonucleotide comprising at least ten nucleotides complementary to WAF1 gene to said cells to inhibit the expression of a WAF1 gene.    
     
     
         29 . A method for promoting growth of cells, comprising the step of: 
 administering to said cells to inhibit the expression of WAF1, an antisense WAF1 construct comprising:    a. a transcriptional promoter;    b. a transcriptional terminator;    c. a DNA segment comprising one or more segments of the WAF1 gene, said gene segment located between said promoter and said terminator, said DNA segment being inverted with respect to said promoter and said terminator, whereby RNA produced by transcription of the DNA segment is complementary to a corresponding segment of WAF1 RNA produced by human cells.    
     
     
         30 . The method of  claim 29  wherein said transcriptional promoter is inducible.  
     
     
         31 . A method for assessing susceptibility to cancers, comprising the step of: 
 testing a tissue selected from the group consisting of blood, chorionic villi, amniotic fluid, and a blastomere of a preimplantation embryo, to determine if DNA in said tissue contains a mutant WAF1 gene.