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p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System

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 expre...

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Autores principales: Andreotti, Virginia, Ciribilli, Yari, Monti, Paola, Bisio, Alessandra, Lion, Mattia, Jordan, Jennifer, Fronza, Gilberto, Menichini, Paola, Resnick, Michael A., Inga, Alberto
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3107237/
https://www.ncbi.nlm.nih.gov/pubmed/21674059
http://dx.doi.org/10.1371/journal.pone.0020643
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author Andreotti, Virginia
Ciribilli, Yari
Monti, Paola
Bisio, Alessandra
Lion, Mattia
Jordan, Jennifer
Fronza, Gilberto
Menichini, Paola
Resnick, Michael A.
Inga, Alberto
author_facet Andreotti, Virginia
Ciribilli, Yari
Monti, Paola
Bisio, Alessandra
Lion, Mattia
Jordan, Jennifer
Fronza, Gilberto
Menichini, Paola
Resnick, Michael A.
Inga, Alberto
author_sort Andreotti, Virginia
collection PubMed
description 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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spelling pubmed-31072372011-06-13 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 PLoS One Research Article 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. Public Library of Science 2011-06-02 /pmc/articles/PMC3107237/ /pubmed/21674059 http://dx.doi.org/10.1371/journal.pone.0020643 Text en This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
spellingShingle Research Article
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
title p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System
title_full p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System
title_fullStr p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System
title_full_unstemmed p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System
title_short p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System
title_sort p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3107237/
https://www.ncbi.nlm.nih.gov/pubmed/21674059
http://dx.doi.org/10.1371/journal.pone.0020643
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