p53 Mutants Have Selective Dominant-Negative Effects on Apoptosis but Not Growth Arrest in Human Cancer Cell Lines

The tumor suppressor gene p53 has been the subject of intense scientific inquiry due to the high frequency of mutations in human cancers (23, 27). A number of functions have been attributed to the wild-type p53 protein. Introduction of exogenous wild-type p53 into cancer cells and its subsequent overexpression result in growth arrest (3, 17, 19, 26). This growth arrest was initially shown to occur at the G1/S cell cycle checkpoint (31). This phenotype is most likely due to the fact that p53 acts as a sequence-specific transactivator of genes containing a p53 consensus binding site in their promoters (16, 40). The chief mediator of p53-mediated G1 arrest was shown to be the p21WAF1/CIP1 gene, a cyclin–cyclin-dependent kinase complex inhibitor (14, 24, 46). In some cell types, introduction and overexpression of wild-type p53 resulted in apoptosis (43, 49). Thus, wild-type p53 acts as a tumor suppressor by causing a cell to either arrest growth or undergo apoptosis following activation by DNA-damaging events (28, 31, 43, 49). Although this differential response to wild-type p53 overexpression would seem to be a cell-type-specific effect, recent studies indicate that specific domains and even specific residues of p53 may allow it to work in combination with the cellular milieu to bring about either a growth arrest or an apoptotic response (1, 21, 25, 32, 41). Interestingly, it has become clearer that some p53 mutations affect apoptosis but not growth arrest (25) or vice versa (1, 21, 32, 41). This has led to a model in which p53-mediated growth arrest and apoptotic pathways are regulated differently. These previous studies have also given further insight into the mechanism by which a cell that has only mutant p53 protein at its disposal is unable to elicit its tumor-suppressive effects. The question arises as to what happens to the growth arrest and apoptosis pathways when mutant and wild-type p53 proteins are expressed in the same cell. Given that p53 binds to its DNA target sequences as a tetramer (11) and that a majority of p53 mutations found in human cancer are missense mutations (27), most mutant p53 monomers are capable of oligomerizing with wild-type p53 monomers. Moreover, the chance that heterotetramer formation occurs at steady-state conditions is probably increased due to the increased half-life of mutant p53 compared to wild-type p53 within a cell (18). Initial in vitro experiments in which mutant p53 was coexpressed with wild-type p53 indicated that the resulting heteromer adopted a mutant conformation (35). Investigations of this dominant-negative effect in vivo have yielded conflicting results. For nearly all mutants studied, dominant-negative effects are seen only when expression of the mutant p53 protein is in excess over wild type. Measurements of this phenomenon were primarily with transactivation assays using a consensus p53 binding site-luciferase-chloramphenicol acetyltransferase reporter construct (29, 39). One study which examined both transactivation and growth arrest parameters found that dominant-negative effects were seen in transactivation assays but not in growth arrest assays (20), indicating the importance of using several different assays to determine dominant-negative effects. In the study reported here, we sought to determine whether there were differential dominant-negative effects on transcription regulation, growth arrest, and apoptosis with the use of a novel bidirectional expression vector. This system provides for the equal expression of wild-type and mutant p53 transcripts in the same cell. We found that dominant-negative effects are seen with only a subset of the p53 missense mutants examined and that this dominant-negative effect was manifested in the apoptotic process but not on growth arrest. Furthermore, although dominant-negative efforts were observed in Bax transactivation assays, these did not all correspond to dominant-negative apoptotic effects.