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

Luc-Akt-PH/YFP Stable MCF-7/B2 Stable Cell Line express the pleckstrin homology (PH) domain of Akt fused to Renilla luciferase with a YFP-Mem, allowing the ability to monitor the PIP3 production in real time through the established BRET biosensor pair. In which the Luc-Akt-PH will be recruited to the plasma membrane due to PIP3 production which then results in an energy transfer between the luciferase and YFP. The cells are suitable for the screening and study of molecules on the PI3K/Akt pathway for diabetes and cancer-related research. This cell line is the can be used with the Luc-Akt-PH Stable MCF7 Cell Line as a control

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 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 for BRET experiments on the following day. This cell line is capable of responding to different growth factors, and that 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 could 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