The human tumor suppressor protein and transcription factor p53 integrates stress signals, such as DNA damage, hypoxia and ribonucleotide depletion, and induces either cell cycle arrest or apoptosis. Approximately 50 percent of all human cancers carry p53 gene mutations. Another 5 to 10 percent inactivate the p53 protein directly by overexpression of high-risk human papilloma virus (HPV) E6 or MDM2, a physiological negative regulator of p53. Considering the plethora of upstream signals, all using different mechanisms of p53 activation, and of p53 downstream effector genes, it is very likely that the remaining human cancers also directly inactivate p53 protein by overexpressing currently unknown negative regulators of p53. We devised a genetic assay for p53 in S. cerevisiae, called the "p53 dissociator assay", to select for such, and other, proteins important for p53 biology in highly efficient screens. We showed that SV40 large T antigen, a known negative regulator, scores in this assay, and we recreated the complex interplay of p53, E6-associated protein (E6-AP) and high-risk HPV E6 resulting in p53 degradation. Three p53 dissociator screens yielded MDM2 and high-risk HPV E6, two established negative regulators, 53BP1, a coactivator of p53, and 23 candidate proteins without current connections to p53 biology. Besides negative and positive regulators of p53, we may also have isolated proteins negatively regulated by p53 in human cells, as suggested by their biology. All three classes of proteins represent important drug targets for improved cancer therapies: inhibition of negative regulators will unleash p53 and induce apoptosis in cancer cells, studies of p53 enhancers will elucidate new mechanisms of further potentiating p53 activity and proteins negatively regulated by p53 will provide new directions to interfere pharmacologically with unchecked proliferation. We propose to study the set of candidate proteins using secondary yeast and mammalian assays, followed by assays tailored to the classes that confirmed p53 dissociators likely belong to. Novel negative regulators of p53 will be pursued further by checking for their overexpression in cancer cell lines with wild-type p53 genes and by setting up reverse two-hybrid assays to screen for drugs able to abrogate their effect on p53. Several specific ways of direct p53 protein inactivation exist, but their exact mechanisms have not been elucidated: cytoplasmic sequestration, p53 degradation involving the human protein E6-AP and nuclear sequestration in teratocarcinoma cell lines. To identify the proteins underlying these mechanisms, we will perform additional targeted D53 dissociator screens in the presence of E6-A P and using specific cDNA libraries.