Cell regulatory genes, encoded products, and uses related thereto

Abstract

This application describes the cloning of p63, a gene at chromosome 3 q 27-29, that bears homology to the tumor suppressor p53. The p63 gene encodes at least six different isotypes. p63 was detected in a variety of human and mouse tissue and demonstrates remarkably divergent activities, such as the ability to transactivate p53 reporter genes and induce apoptosis. Isotopes of p63 lacking a transactivation domain act as dominant negatives towards the transactivation by p53 and p63.

We claim:  
     
         1 . An isolated antibody that binds to a p63 protein and does not detectably bind to a p53 or p73 protein.  
     
     
         2 . The antibody of  claim 1 , wherein the antibody is specifically immunoreactive with a p63 protein.  
     
     
         3 . The antibody of  claim 1 , wherein the antibody binds to a p63 protein comprising an amino acid sequence having at least about 90% identity with a sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         4 . The antibody of  claim 3 , wherein the antibody binds to a p63 protein comprising an amino acid sequence having at least about 95% identity with a sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         5 . The antibody of  claim 4 , wherein the antibody binds to a p63 protein comprising an amino acid sequence having at least about 98% identity with a sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         6 . The antibody of  claim 3 , wherein the antibody binds to a p63 protein comprising an amino acid sequence having a sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         7 . The antibody of  claim 1 , wherein the antibody binds to a p63 protein encoded by a nucleic acid sequence which hybridizes under stringent conditions to the complementary strand of a nucleic acid having a sequence set forth in any one of SEQ ID NOs: 1-12.  
     
     
         8 . The antibody of  claim 7 , wherein the antibody binds to a p63 protein encoded by a nucleic acid sequence of any one of SEQ ID NOs: 1-12.  
     
     
         9 . The antibody of  claim 1 , wherein the antibody binds to a p63 protein comprising the amino acid sequence set forth in SEQ ID NO: 16.  
     
     
         10 . The antibody of  claim 1 , wherein binding is determined using immunoprecipitation, immunostaining, western blotting, or a radioimmunoassay (RIA).  
     
     
         11 . The antibody of  claim 1 , wherein the antibody is a monoclonal antibody.  
     
     
         12 . The antibody of  claim 1 , wherein the antibody is a recombinant antibody.  
     
     
         13 . The antibody of  claim 1 , wherein the antibody is an antibody fragment.  
     
     
         14 . The antibody of  claim 13 , wherein the antibody fragment is an Fab, F(ab′) 2 , Fab′, Fv, or scFv.  
     
     
         15 . The antibody of  claim 1 , wherein the antibody is a mouse antibody.  
     
     
         16 . The antibody of  claim 1 , wherein the antibody is a human antibody.  
     
     
         17 . The antibody of  claim 1 , wherein the antibody is a humanized antibody.  
     
     
         18 . The antibody of  claim 1 , wherein the antibody comprises a label.  
     
     
         19 . The antibody of  claim 18 , wherein the label is at least one of the following: a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.  
     
     
         20 . The antibody of  claim 1 , wherein the antibody is antibody 4A4.  
     
     
         21 . The antibody of  claim 1 , wherein the antibody binds to the epitope to which antibody 4A4 binds.  
     
     
         22 . The antibody of  claim 1 , wherein the antibody inhibits binding of p63 to antibody 4A4.  
     
     
         23 . An isolated antibody that selectively binds to a p63 protein relative to p53 and p73 proteins.  
     
     
         24 . A hybridoma producing the antibody of  claim 11 .  
     
     
         25 . A purified preparation of polyclonal antibodies wherein the antibodies bind to a p63 protein and do not detectably bind to a p53 or p73 protein.  
     
     
         26 . The purified preparation of  claim 25 , wherein the antibodies are specifically immunoreactive with a p63 protein.  
     
     
         27 . The purified preparation of  claim 25 , wherein the antibodies are antibody fragments.  
     
     
         28 . The antibody of  claim 25 , wherein binding is determined using immunoprecipitation, immunostaining, western blotting, or a radioimmunoassay (RIA).  
     
     
         29 . An isolated polypeptide wherein said polypeptide comprises (a) an amino acid sequence set forth in any one of SEQ ID NOs: 13-24; (b) an amino acid sequence having at least about 90% identity with an amino acid sequence set forth in any one of SEQ ID NOs: 13-24; (c) an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to the complementary strand of a nucleic acid having a sequence set forth in any one of SEQ ID NOs: 1-12; or (d) a fragment of an amino acid sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         30 . The isolated polypeptide of  claim 29 , wherein said polypeptide has one or more of the following biological activities: (i) binds a target DNA sequence, (ii) transactivates a target gene, (iii) induces apoptosis, (iv) oligomerizes, (v) localizes to basal epithelial cells, or (vi) localizes to squamous cervical cells.  
     
     
         31 . The isolated polypeptide of  claim 30 , wherein said target DNA sequence is at least one of the following: (i) a p53-responsive element, (ii) a minimal p53 target binding sequence, or (iii) a p53 target DNA binding sequence in a p21 promoter.  
     
     
         32 . The isolated polypeptide of  claim 29 , wherein said polypeptide is a fusion protein.  
     
     
         33 . The isolated polypeptide of  claim 32 , wherein said polypeptide is functional in a two-hybrid assay.  
     
     
         34 . The isolated polypeptide of  claim 29 , wherein said polypeptide is functions either as an agonist of cell cycle regulation or an antagonist of cell cycle regulation.  
     
     
         35 . The isolated polypeptide of  claim 29 , wherein said polypeptide comprises an amino acid sequence having at least about 95% identity with an amino acid sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         36 . The isolated polypeptide of  claim 35 , wherein said polypeptide comprises an amino acid sequence having at least about 98% identity with an amino acid sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         37 . The isolated polypeptide of  claim 29 , wherein said polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         38 . The isolated polypeptide of  claim 29 , wherein said polypeptide is a mammalian polypeptide.  
     
     
         39 . The isolated polypeptide of  claim 38 , wherein said polypeptide is a human polypeptide.  
     
     
         40 . A kit for detecting a p63 protein comprising (i) an isolated anti-p63 antibody, or fragment thereof, and (ii) a means for detecting the anti-p63 antibody.  
     
     
         41 . The kit of  claim 40 , wherein the means for detecting the anti-p63 antibody is one or more of a buffer, formaldehyde, an enzyme, a co-enzyme, a substrate, a polypeptide, an antibody, or a detectable label.  
     
     
         42 . The kit of  claim 41 , wherein the means for detecting the anti-p63 antibody is a detectable label conjugated to the anti-p63 antibody.  
     
     
         43 . The kit of  claim 41 , wherein means for detecting the anti-p63 antibody is a second antibody immunoreactive with the anti-p63 antibody.  
     
     
         44 . The kit of  claim 40 , wherein the anti-p63 antibody binds to a p63 protein and does not detectably bind to a p53 or p73 protein.  
     
     
         45 . The kit of  claim 44 , wherein the anti-p63 antibody is specifically immunoreactive with a p63 protein.  
     
     
         46 . The kit of  claim 40 , wherein the anti-p63 antibody binds to a p63 protein comprising an amino acid sequence having at least about 90% identity with a sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         47 . The kit of  claim 40 , wherein the anti-p63 antibody binds to a p63 protein comprising an amino acid sequence having a sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         48 . The kit of  claim 40 , wherein the anti-p63 antibody is a monoclonal antibody.  
     
     
         49 . The kit of  claim 40 , wherein the anti-p63 antibody is a purified preparation of polyclonal antibodies.  
     
     
         50 . The kit of  claim 40 , wherein the anti-p63 antibody is provided in a form suitable for detecting a p63 protein in cells.  
     
     
         51 . A kit for immunostaining a sample of cells, comprising: one or more antibodies, or fragments thereof, for selectively binding p63 protein in the sample, relative to p53 and p73; and a means for selectively detecting the antibody bound to p63 protein in the sample of cells.  
     
     
         52 . The kit of  claim 51 , further comprising one or more reagents for fixing said sample of cells before immunostaining.  
     
     
         53 . The kit of  claim 51 , wherein the sample of cells is a tissue section.  
     
     
         54 . The kit of  claim 51 , wherein the means for selectively detecting the antibody is an agent that binds to the antibody and which includes a label that is detectable by spectrophotometry or fluorometry.  
     
     
         55 . The kit of  claim 51 , wherein the means for selectively detecting the antibody is an agent that binds to the antibody and which includes an enzyme that acts on a chromogenic substrate.  
     
     
         56 . The kit of  claim 51 , wherein the means for selectively detecting the antibody is a label that is detectable by spectrophotometry or fluorometry, or an enzyme that acts on a chromogenic substrate, which label or enzyme is conjugated to the antibody.  
     
     
         57 . The kit of  claim 51 , wherein the antibody binds to a p63 protein having an amino acid sequence set forth in any one of SEQ ID NOs: 13-24.  
     
     
         58 . The kit of  claim 51 , wherein the antibody is a monoclonal antibody.  
     
     
         59 . The kit of  claim 51 , wherein the one or more antibodies is a preparation of p63-specific polyclonal antibodies.  
     
     
         60 . The kit of  claim 51 , wherein the antibody differentially binds for different isotypes of p63 proteins.  
     
     
         61 . The kit of  claim 51 , wherein the antibody binds to one or more p63 isotypes selected from the group consisting of TA*p63, TAp63, and ΔNp63.  
     
     
         62 . A kit for determining the level of p63 protein in a sample, comprising: an antibody, or fragment thereof, which is selectively immunoreactive with a p63 protein, relative to p53 and p73 proteins; and a control protein including a p63 peptide sequence that is bound by the antibody.