P53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system

Virginia Andreotti, Yari Ciribilli, Paola Monti, Alessandra Bisio, Mattia Lion, Jennifer Jordan, Gilberto Fronza, Paola Menichini, Michael A. Resnick, Alberto Inga

Research output: Contribution to journalArticle

29 Citations (Scopus)

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. Methodology/Principal Findings: 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/Significance: 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.

Original languageEnglish
Article numbere20643
JournalPLoS One
Volume6
Issue number6
DOIs
Publication statusPublished - 2011

Fingerprint

transcriptional activation
Yeast
Transcriptional Activation
Screening
Yeasts
yeasts
screening
mutation
Mutation
Molecules
neoplasms
Tumors
luciferase
Neoplasms
Proteins
Luciferases
proteins
mutants
Assays
ABC transporters

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

P53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system. / Andreotti, Virginia; Ciribilli, Yari; Monti, Paola; Bisio, Alessandra; Lion, Mattia; Jordan, Jennifer; Fronza, Gilberto; Menichini, Paola; Resnick, Michael A.; Inga, Alberto.

In: PLoS One, Vol. 6, No. 6, e20643, 2011.

Research output: Contribution to journalArticle

Andreotti, Virginia ; Ciribilli, Yari ; Monti, Paola ; Bisio, Alessandra ; Lion, Mattia ; Jordan, Jennifer ; Fronza, Gilberto ; Menichini, Paola ; Resnick, Michael A. ; Inga, Alberto. / P53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system. In: PLoS One. 2011 ; Vol. 6, No. 6.
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AB - 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. Methodology/Principal Findings: 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/Significance: 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.

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