The p53 tumor suppressor gene induces cell cycle arrest and/or apoptosis when activated by DNA damage. Because cancer is treated with DNA- damaging agents, the outcome of therapy is significantly influenced by the ability of p53 to induce apoptosis in response to DNA damage. Thus, tumors that express wild-type p53 tend to respond better to therapy and are more radiosensitive than tumors expressing mutant inactive p53. One exception is melanoma; these tumors express wild-type p53, but are extremely radioresistant. Our goal is to understand at the molecular level why wild-type p53 does not induce apoptosis of melanoma cells with damaged DNA. In normal cells, p53-dependent apoptosis in response to DNA damage occurs in three steps; a) increase in the half-life of the p53 protein, leading to increased protein levels, and increase in the affinity of p53 for specific DNA sequences; b) binding of p53 to regulatory regions of specific genes and induction of transcription of these genes; c) induction of apoptosis by the protein products of the p53-inducible genes. Our preliminary results suggest that in melanoma cells, p53, even in the absence of DNA damage, has constitutively high affinity for DNA. We also find a defect in the ability of p53 to activate transcription of specific target genes, such as mdm2 gene. We therefore hypothesize that the radioresistance of melanoma is due to two molecular events that occur during transformation of melanocytes to melanoma cells; a) an aberration in the DNA damage response leading to constitutive activation of p53; and b) a defect in the ability of p53 to function as a transcription factor for the full repertoire of p53-target genes, allowing melanoma cells to escape apoptosis. To address this hypothesis we will: 1) analyze the aberrant response of p53 to DNA damage during melanoma progression and determine whether it can be used as a diagnostic and prognostic marker; 2) determine whether a defect in the ability of p53 to induce expression of the full repertoire of p53-target genes underlies the escape of melanoma cells from p53-dependent apoptosis; and 3 determine whether altered DNA binding sequence-specificity and/or comprised transcriptional activity underlie the inability of p53 to induce expression of the full repertoire of p53-target genes. These experiments will help identify therapeutic strategies aimed at restoring apoptosis to melanoma cells.