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A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells

High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of malignant transformation in pre-neoplastic lesions. Targeting t...

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Autores principales: Messa, Lorenzo, Celegato, Marta, Bertagnin, Chiara, Mercorelli, Beatrice, Nannetti, Giulio, Palù, Giorgio, Loregian, Arianna
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902497/
https://www.ncbi.nlm.nih.gov/pubmed/29662081
http://dx.doi.org/10.1038/s41598-018-24470-4
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author Messa, Lorenzo
Celegato, Marta
Bertagnin, Chiara
Mercorelli, Beatrice
Nannetti, Giulio
Palù, Giorgio
Loregian, Arianna
author_facet Messa, Lorenzo
Celegato, Marta
Bertagnin, Chiara
Mercorelli, Beatrice
Nannetti, Giulio
Palù, Giorgio
Loregian, Arianna
author_sort Messa, Lorenzo
collection PubMed
description High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of malignant transformation in pre-neoplastic lesions. Targeting the viral E6 oncoprotein through inhibitory compounds can counteract the survival of cancer cells due to the reactivation of p53-mediated pathways and represents an intriguing strategy to treat HPV-associated neoplasias. Here, we describe the development of a quantitative and easy-to-perform assay to monitor the E6-mediated degradation of p53 in living cells to be used for small-molecule testing. This assay allows to unbiasedly determine whether a compound can protect p53 from the E6-mediated degradation in cells, through a simple 3-step protocol. We validated the assay by testing two small molecules, SAHA and RITA, reported to impair the E6-mediated p53 degradation. Interestingly, we observed that only SAHA efficiently rescued p53, while RITA could not provide the same degree of protection. The possibility to specifically and quantitatively monitor the ability of a selected compound to rescue p53 in a cellular context through our LumiFluo assay could represent an important step towards the successful development of anti-HPV drugs.
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spelling pubmed-59024972018-04-25 A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells Messa, Lorenzo Celegato, Marta Bertagnin, Chiara Mercorelli, Beatrice Nannetti, Giulio Palù, Giorgio Loregian, Arianna Sci Rep Article High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of malignant transformation in pre-neoplastic lesions. Targeting the viral E6 oncoprotein through inhibitory compounds can counteract the survival of cancer cells due to the reactivation of p53-mediated pathways and represents an intriguing strategy to treat HPV-associated neoplasias. Here, we describe the development of a quantitative and easy-to-perform assay to monitor the E6-mediated degradation of p53 in living cells to be used for small-molecule testing. This assay allows to unbiasedly determine whether a compound can protect p53 from the E6-mediated degradation in cells, through a simple 3-step protocol. We validated the assay by testing two small molecules, SAHA and RITA, reported to impair the E6-mediated p53 degradation. Interestingly, we observed that only SAHA efficiently rescued p53, while RITA could not provide the same degree of protection. The possibility to specifically and quantitatively monitor the ability of a selected compound to rescue p53 in a cellular context through our LumiFluo assay could represent an important step towards the successful development of anti-HPV drugs. Nature Publishing Group UK 2018-04-16 /pmc/articles/PMC5902497/ /pubmed/29662081 http://dx.doi.org/10.1038/s41598-018-24470-4 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Messa, Lorenzo
Celegato, Marta
Bertagnin, Chiara
Mercorelli, Beatrice
Nannetti, Giulio
Palù, Giorgio
Loregian, Arianna
A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells
title A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells
title_full A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells
title_fullStr A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells
title_full_unstemmed A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells
title_short A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells
title_sort quantitative lumifluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein e6 in living cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902497/
https://www.ncbi.nlm.nih.gov/pubmed/29662081
http://dx.doi.org/10.1038/s41598-018-24470-4
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