A breast-cancer cell line expressing a BRET biosensor to monitor PI-3 kinase activity in real time

We have developed a MCF7-derived cell line stably co-expressing the pleckstrin homology (PH) domain of Akt fused to Renilla luciferase (Luc-Akt-PH) and a plasma membrane-targeted YFP (YFP-Mem). Upon ligand stimulation, PIP3 production by PI-3 kinase induces the recruitment of Luc-Akt-PH at the plasma membrane, resulting in an energy transfer between Luciferase and the membrane-targeted YFP (Fig. 1). This permits to monitor PIP3 production in real time, in living cells. These cells are suitable for the screening and study of the effects of ligands and drugs on the PI3K/Akt pathway, notably in the context of diabetes and cancer-related research.

Interest / Relevance: The PI3K (phosphatidylinositol 3-kinase)/Akt pathway regulates multiple biological processes such as metabolism, cell proliferation, survival, migration and apoptosis. This pathway is therefore an important target for drug discovery. However, current assays for measurement of PIP3 production are technically demanding and not amenable to high-throughput screening.
We have developed a cell line (MCF-7/B2 cells) expressing a BRET-based biosensor that permits to monitor, in real time, in living cells, ligand-induced PIP3 production at the plasma membrane.
After seeding in 96 wells plates, cells are ready to use on the following day for BRET experiments. This cell line is capable of responding to different hormones and growth factors. Moreover, the effects of known inhibitors of the PI3K/Akt pathway can be readily detected, indicating that this stable clone is suitable for screening and/or studying inhibitors or activators of the PI3K/Akt pathway. This tool also allows for the study of PI3K-stimulatory activity present in biological fluids (e.g. serum, plasma/u2026).
This cell line therefore constitutes a powerful tool for the study of activators and inhibitors acting at different levels on this signaling pathway. MCF-7/B2 cells can also be used in high-throughput screening assays for the identification of new molecules that modulate the PI3K/Akt pathway.

Keywords: Cancer, Diabetes, Tyrosine kinase signaling, PI-3 kinase/Akt pathway, Phosphatidyl Inosintol 3 phosphate

Scientist's name: Tarik ISSAD

Publications:

Development of a human breast-cancer derived cell line stably expressing a bioluminescence resonance energy transfer (BRET)-based phosphatidyl inositol-3 phosphate (PIP3) biosensor.

Kuo MS, Auriau J, Pierre-Eugène C, Issad T.

PLoS One. 2014 Mar 19,9(3):e92737. doi: 10.1371/journal.pone.0092737. eCollection 2014.

PMID: 24647478

Reference:

RT00733