The PI3 Kinase/AKT pathway plays a critical role in cell survival signaling and the activation of this pathway has been linked to tumorigenesis. Upregulation of AKT and PI3 Kinase is seen in many tumor types and the negative regulator of this pathway PTEN is deleted in a number of tumor types. The Forkhead family of transcription factors (FKHR, FKHRL1, and AFX) are downstream targets of the PI3 Kinase/AKT pathway. Phosphorylation of the Forkhead transcription factors by AKT results in their cytoplasmic retention and inactivation, inhibiting the expression of Forkhead regulated genes which control the cell cycle, cell death and oxidative stress allowing cell proliferation and inhibition of apoptosis. Therefore this pathway remains an attractive target for cancer drug discovery. We developed a medium throughput assay to evaluate the cell-based activity of PI3 Kinase/AKT pathway inhibitors quantitating the phosphorylation level of AFX. We generated a U87MG glioblastoma cell line stably overexpressing an AFX-GFP (green fluorescent protein) and characterized it to confirm that the phosphorylation, nuclear translocation AFX-GFP fusion protein remains intact. Then we treated these cells with PI3 Kinase/AKT pathway inhibitors in a 96-well format and quantitated the phosphorylation status of the AFX-GFP fusion using a novel sandwich ELISA. The ELISA analysis showed that PI3 Kinase inhibitor Wortmannin inhibits AFX-GFP phosphorylation in these cell lines with an IC50 of 400 nM. Other pathway inhibitors such as Staurosporine, LY 294002, also inhibit the AFX-GFP phosphorylation activity. This ELISA was utilized to rapidly evaluate the cellular activity of PI3 Kinase/AKT pathway inhibitors as potential cancer therapeutic agents.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]