title: p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system.
abstract: BACKGROUND
  The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4.
  
  
  RESULTS
  Given the many factors that might influence p53 function, including expression levels, mutations, cofactor proteins and small molecules, we expanded our previously described yeast-based system to provide the opportunity for efficient investigation of their individual and combined impacts in a miniaturized format. The system integrates i) variable expression of p53 proteins under the finely tunable GAL1,10 promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1.
  
  
  CONCLUSIONS
  We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins.
authors.affiliation: Unit of Molecular Mutagenesis, National Institute for Cancer Research, IST, Genoa, Italy.
authors.forename: Virginia
authors.initials: V
authors.lastname: Andreotti
authors.forename: Yari
authors.initials: Y
authors.lastname: Ciribilli
authors.forename: Paola
authors.initials: P
authors.lastname: Monti
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bisio
authors.forename: Mattia
authors.initials: M
authors.lastname: Lion
authors.forename: Jennifer
authors.initials: J
authors.lastname: Jordan
authors.forename: Gilberto
authors.initials: G
authors.lastname: Fronza
authors.forename: Paola
authors.initials: P
authors.lastname: Menichini
authors.forename: Michael A
authors.initials: MA
authors.lastname: Resnick
authors.forename: Alberto
authors.initials: A
authors.lastname: Inga
mesh_terms: D000818:Animals; D017930:Genes, Reporter / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D049410:Luciferases, Renilla / Q000235:genetics; D009154:Mutation; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D015533:Transcriptional Activation / Q000187:drug effects* / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism*; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; C498301:TP53BP1 protein, human; D016159:Tumor Suppressor Protein p53; D000071857:Tumor Suppressor p53-Binding Protein 1; D049410:Luciferases, Renilla; D051736:Proto-Oncogene Proteins c-mdm2