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Development of a Human Breast-Cancer Derived Cell Line Stably Expressing a Bioluminescence Resonance Energy Transfer (BRET)-Based Phosphatidyl Inositol-3 Phosphate (PIP(3)) Biosensor
Stimulation of tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP(2) to generate PIP(3), which recruit Akt to the plasma membrane through its pleckstrin homology (PH) domain, permitting its activation by PDKs. Activated Akt controls important biologi...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960261/ https://www.ncbi.nlm.nih.gov/pubmed/24647478 http://dx.doi.org/10.1371/journal.pone.0092737 |
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author | Kuo, Mei-Shiue Auriau, Johanna Pierre-Eugène, Cécile Issad, Tarik |
author_facet | Kuo, Mei-Shiue Auriau, Johanna Pierre-Eugène, Cécile Issad, Tarik |
author_sort | Kuo, Mei-Shiue |
collection | PubMed |
description | Stimulation of tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP(2) to generate PIP(3), which recruit Akt to the plasma membrane through its pleckstrin homology (PH) domain, permitting its activation by PDKs. Activated Akt controls important biological functions, including cell metabolism, proliferation and survival. The PI3K pathway is therefore an attractive target for drug discovery. However, current assays for measurement of PIP(3) production are technically demanding and not amenable to high-throughput screening. We have established a MCF-7-derived breast cancer cell line, that stably co-expresses the PH domain of Akt fused to Renilla luciferase and YFP fused to a membrane localization signal. This BRET biosensor pair permits to monitor, in real time, in living cells, PIP(3) production at the plasma membrane upon stimulation by different ligands, including insulin, the insulin analogue glargine, IGF1, IGF2 and EGF. Moreover, several known inhibitors that target different steps of the PI3K/Akt pathway caused inhibition of ligand-induced BRET. Cetuximab, a humanized anti-EGF receptor monoclonal antibody used for the treatment of cancer, completely inhibited EGF-induced BRET, and the tyrosine kinase inhibitor tyrphostine AG1024 inhibited insulin effect on PIP(3) production. Moreover, the effects of insulin and IGF1 were inhibited by molecules that inhibit PI3K catalytic activity or the interaction between PIP(3) and the PH domain of Akt. Finally, we showed that human serum induced a dose-dependent increase in BRET signal, suggesting that this stable clone may be used as a prognostic tool to evaluate the PI3K stimulatory activity present in serum of human patients. We have thus established a cell line, suitable for the screening and/or the study of molecules with stimulatory or inhibitory activities on the PI3K/Akt pathway that will constitute a new tool for translational research in diabetes and cancer. |
format | Online Article Text |
id | pubmed-3960261 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-39602612014-03-24 Development of a Human Breast-Cancer Derived Cell Line Stably Expressing a Bioluminescence Resonance Energy Transfer (BRET)-Based Phosphatidyl Inositol-3 Phosphate (PIP(3)) Biosensor Kuo, Mei-Shiue Auriau, Johanna Pierre-Eugène, Cécile Issad, Tarik PLoS One Research Article Stimulation of tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP(2) to generate PIP(3), which recruit Akt to the plasma membrane through its pleckstrin homology (PH) domain, permitting its activation by PDKs. Activated Akt controls important biological functions, including cell metabolism, proliferation and survival. The PI3K pathway is therefore an attractive target for drug discovery. However, current assays for measurement of PIP(3) production are technically demanding and not amenable to high-throughput screening. We have established a MCF-7-derived breast cancer cell line, that stably co-expresses the PH domain of Akt fused to Renilla luciferase and YFP fused to a membrane localization signal. This BRET biosensor pair permits to monitor, in real time, in living cells, PIP(3) production at the plasma membrane upon stimulation by different ligands, including insulin, the insulin analogue glargine, IGF1, IGF2 and EGF. Moreover, several known inhibitors that target different steps of the PI3K/Akt pathway caused inhibition of ligand-induced BRET. Cetuximab, a humanized anti-EGF receptor monoclonal antibody used for the treatment of cancer, completely inhibited EGF-induced BRET, and the tyrosine kinase inhibitor tyrphostine AG1024 inhibited insulin effect on PIP(3) production. Moreover, the effects of insulin and IGF1 were inhibited by molecules that inhibit PI3K catalytic activity or the interaction between PIP(3) and the PH domain of Akt. Finally, we showed that human serum induced a dose-dependent increase in BRET signal, suggesting that this stable clone may be used as a prognostic tool to evaluate the PI3K stimulatory activity present in serum of human patients. We have thus established a cell line, suitable for the screening and/or the study of molecules with stimulatory or inhibitory activities on the PI3K/Akt pathway that will constitute a new tool for translational research in diabetes and cancer. Public Library of Science 2014-03-19 /pmc/articles/PMC3960261/ /pubmed/24647478 http://dx.doi.org/10.1371/journal.pone.0092737 Text en © 2014 Kuo et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Kuo, Mei-Shiue Auriau, Johanna Pierre-Eugène, Cécile Issad, Tarik Development of a Human Breast-Cancer Derived Cell Line Stably Expressing a Bioluminescence Resonance Energy Transfer (BRET)-Based Phosphatidyl Inositol-3 Phosphate (PIP(3)) Biosensor |
title | Development of a Human Breast-Cancer Derived Cell Line Stably Expressing a Bioluminescence Resonance Energy Transfer (BRET)-Based Phosphatidyl Inositol-3 Phosphate (PIP(3)) Biosensor |
title_full | Development of a Human Breast-Cancer Derived Cell Line Stably Expressing a Bioluminescence Resonance Energy Transfer (BRET)-Based Phosphatidyl Inositol-3 Phosphate (PIP(3)) Biosensor |
title_fullStr | Development of a Human Breast-Cancer Derived Cell Line Stably Expressing a Bioluminescence Resonance Energy Transfer (BRET)-Based Phosphatidyl Inositol-3 Phosphate (PIP(3)) Biosensor |
title_full_unstemmed | Development of a Human Breast-Cancer Derived Cell Line Stably Expressing a Bioluminescence Resonance Energy Transfer (BRET)-Based Phosphatidyl Inositol-3 Phosphate (PIP(3)) Biosensor |
title_short | Development of a Human Breast-Cancer Derived Cell Line Stably Expressing a Bioluminescence Resonance Energy Transfer (BRET)-Based Phosphatidyl Inositol-3 Phosphate (PIP(3)) Biosensor |
title_sort | development of a human breast-cancer derived cell line stably expressing a bioluminescence resonance energy transfer (bret)-based phosphatidyl inositol-3 phosphate (pip(3)) biosensor |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960261/ https://www.ncbi.nlm.nih.gov/pubmed/24647478 http://dx.doi.org/10.1371/journal.pone.0092737 |
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