Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer. A majority of these mutations are missense mutations in the DNAbinding domain. As a result, the mutated p53 gene encodes a full-length protein incapable of transactivating its target genes. In addition to this loss of function, mutant p53 can have a dominant negative effect over wild-type p53 and/or gain of function activity independently of the wild-type protein.To better understand the nature of the tumorigenic activity of mutant p53, we have investigated the mechanism by which mutant p53 can exert a dominant negative effect. We have established several stable cell lines capable of inducibly expressing a p53 mutant alone, wild-type p53 alone, or both proteins concurrently. In this context, we have used chromatin immunoprecipitation to determine the ability of wild-type p53 to bind to its endogenous target genes in the presence of various p53 mutants. We have found that p53 missense mutants markedly reduce the binding of wild-type p53 to the p53 responsive element in the target genes of p21, MDM2, and PIG3. These findings correlate with the reduced ability of wild-type p53 in inducing these and other endogenous target genes and growth suppression in the presence of mutant p53. We also showed that mutant p53 suppresses the ability of wild-type p53 in inducing cell cycle arrest. This highlights the sensitivity and utility of the dual inducible expression system because in previous studies, p53-mediated cell cycle arrest is not affected by transiently overexpressed p53 mutants. Together, our data showed that mutant p53 exerts its dominant negative activity by abrogating the DNA binding, and subsequently the growth suppression, functions of wild-type p53.

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p53 is a tumor suppressor gene that is mutated in greater than 50% of human cancers. The action of p53 as a tumor suppressor involves inhibition of cell proliferation through cell cycle arrest and/or apoptosis. Loss of p53 function therefore allows the uncontrolled proliferation associated with cancerous cells. While design of most anti-cancer agents has focused on targeting and inactivating cancer promoting targets, such as oncogenes, recent attention has been given to restoring the lost activity of tumor suppressor genes. Because the loss of p53 function is so prevalent in human cancer, this protein is an ideal candidate for such therapy. Several gene therapeutic strategies have been employed in the attempt to restore p53 function to cancerous cells. These approaches include introduction of wild-type p53 into cells with mutant p53; the use of small molecules to stabilize mutant p53 in a wild-type, active conformation; and the introduction of agents to prevent degradation of p53 by proteins that normally target it. In addition, because mutant p53 has oncogenic gain of function activity, several approaches have been investigated to selectively target and kill cells harboring mutant p53. These include the introduction of mutant viruses that cause cell death only in cells with mutant p53 and the introduction of a gene that, in the absence of functional p53, produces a toxic product. Many obstacles remain to optimize these strategies for use in humans, but, despite these, restoration of p53 function is a promising anti-cancer therapeutic approach.

p53, a transcription factor, exerts its tumor suppressor activity by regulating a diverse array of genes involved in the control of the cell cycle, apoptosis, differentiation, and DNA repair. Previously, we and others have found that p53 contains multiple separate functional domains, each of which has a unique contribution to the activity of p53 in inducing cell cycle arrest and apoptosis, probably via differential regulation of target genes. We and others have also found that the p53 family members, that is, p53, p63, and p73, are all capable of inducing cell cycle arrest and apoptosis and regulate both common and unique target genes. Here, we used Affymetrix GeneChip assay and Northern blot analysis to determine whether some known target genes are regulated by various p53 mutants, which are active in inducing cell cycle arrest, apoptosis, or both, and to identify novel target genes regulated by the p53 family. We found that various p53 functional domains control the induction of a target gene, which may be responsible for the unique activity of a given functional domain in inducing cell cycle arrest or apoptosis. In addition, we identified fourteen potential novel target genes that are differentially regulated by various p53 family members. Therefore, the regulation of a known target gene by a defined p53 mutant can be used to classify the role of the target gene in p53 tumor suppression and the identification of these fourteen potential novel target genes of the p53 family can lead to uncover the signaling pathway to which a p53 family member functions in tumor suppression (p53) and in development (p63 and p73).