cDNA encoding P2P proteins and use of P2P cDNA-derived antibodies and antisense reagents in determining the proliferative potential of normal, abnormal, and cancer cells in animals and humans

Abstract

Terminal differentiation is associated with repression in the expression of the P2P subset of hnRNP proteins. The 5173 base pair P2P cDNA was cloned and characterized. The cDNA contains a 4214 base pair open reading frame. Probes to the P2P cDNA detect a single 8 kb mRNA in multiple murine tissues, in proliferating murine 3T3T cells but not in terminally differentiated 3T3T adipocytes. Evidence that the P2P cDNA can encode proteins with domains for hnRNP association was established by showing that the C130 monoclonal antibody, produced against a fusion protein derived from the P2P cDNA, selectively detects native P2P hnRNP proteins. In addition, it was shown that P2P antisense oligonucleotides selectively repressed 30-40 kDa P2P expression. Since terminal differentiation is also associated with modulation in Rb1 function, assays were performed which demonstrated that P2P cDNA products interact with Rb1. Evidence that the P2P cDNA encodes a protein domain that binds Rb1 was established using a GST-P2P fusion protein to selectively precipitate Rb1. Data also show that this binding is competed by the adenovirus E1a protein, indicating that binding occurs through the “pocket” domain of Rb1. These results establish that the P2P cDNA encodes protein domains involved in both hnRNP association and Rb1 binding and complement recent reports that localize Rb1 to sites of RNA processing in the nucleus. The interaction of P2P cDNA products and Rb1 may therefore serve to modulate cell proliferation and/or other biological functions associated with tumor suppression by an RNA processing mechanism.

What is claimed is:  
     
         1 . An isolated nucleic acid sequence which codes for the amino acid sequence shown in FIG. 2.  
     
     
         2 . The isolated nucleic acid sequence of  claim 1  which has the nucleotide sequence as shown from base 139 to base 4353 in FIG. 6.  
     
     
         3 . The isolated nucleic acid sequence of  claim 2  which has the entire nucleic acid sequence shown in FIG. 6.  
     
     
         4 . An isolated ribonucleic acid sequence transcribed from the nucleic acid sequence of  claim 2 .  
     
     
         5 . The ribonucleic acid of  claim 4  which has a nucleotide sequence about 8 kilobases long.  
     
     
         6 . An isolated polypeptide having the amino acid sequence shown in FIG. 2.  
     
     
         7 . The isolated polypeptide of  claim 6  which has a molecular weight of about 160 kDa.  
     
     
         8 . The isolated polypeptide of  claim 6  which consists of 1404 amino acids.  
     
     
         9 . An isolated polypeptide which is encoded by the nucleic acid of  claim 2 .  
     
     
         10 . An isolated antibody which binds to protein products of P2P cDNA.  
     
     
         11 . The isolated antibody of  claim 10  which binds to the carboxy-terminal half of the polypeptide shown in FIG. 2.  
     
     
         12 . The isolated antibody of  claim 11  which is designated C130.  
     
     
         13 . An isolated antisense oligonucleotide which binds to a domain of the open reading frame of  claim 2 .  
     
     
         14 . The isolated antisense oligonucleotide of  claim 13  which has the sequence 5′ CAGCAGGAGCTGTGTT 3′.  
     
     
         15 . A method for repressing the proliferative potential of a cell selected from the group of normal, abnormal, and cancer cells comprising contacting DNA from the cell with an antisense oligonucleotide which binds to a domain of the open reading frame of P2P cDNA.  
     
     
         16 . The method of  claim 15  wherein the antisense oligonucleotide has the sequence 5′ CAGCAGGAGCTGTGTT 3′.