p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system.

<h4>Background</h4>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 occur...

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Autores principales:	Virginia Andreotti, Yari Ciribilli, Paola Monti, Alessandra Bisio, Mattia Lion, Jennifer Jordan, Gilberto Fronza, Paola Menichini, Michael A Resnick, Alberto Inga
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2b8a025dfb6a4609b60f93c77cb8b8e6
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spelling	oai:doaj.org-article:2b8a025dfb6a4609b60f93c77cb8b8e62021-11-18T06:52:41Zp53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system.1932-620310.1371/journal.pone.0020643https://doaj.org/article/2b8a025dfb6a4609b60f93c77cb8b8e62011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21674059/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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.Virginia AndreottiYari CiribilliPaola MontiAlessandra BisioMattia LionJennifer JordanGilberto FronzaPaola MenichiniMichael A ResnickAlberto IngaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 6, p e20643 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Virginia Andreotti Yari Ciribilli Paola Monti Alessandra Bisio Mattia Lion Jennifer Jordan Gilberto Fronza Paola Menichini Michael A Resnick Alberto Inga p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system.
description	<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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.
format	article
author	Virginia Andreotti Yari Ciribilli Paola Monti Alessandra Bisio Mattia Lion Jennifer Jordan Gilberto Fronza Paola Menichini Michael A Resnick Alberto Inga
author_facet	Virginia Andreotti Yari Ciribilli Paola Monti Alessandra Bisio Mattia Lion Jennifer Jordan Gilberto Fronza Paola Menichini Michael A Resnick Alberto Inga
author_sort	Virginia Andreotti
title	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_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_sort	p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/2b8a025dfb6a4609b60f93c77cb8b8e6
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p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system.

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