Abstract #5258: Nonradioactive quantification of phosphoinositide 3-kinase (PI3K) products from breast cancer cell lines

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO Phosphoinositides (PIPs) are an integral part of cell signaling pathways and are metabolized by kinases, phosphatases, and phospholipases. Phosphoinositides are produced by the activities of PI3K and serve as second messengers in cell survival, growth, motility, immune response, inflammation, and apoptosis. Conversely, abnormal production of these signaling lipids leads to proliferative, metabolic, and inflammatory disorders. It is critical to know the absolute and relative concentration of individual phosphoinositides in cell samples, but this analysis is difficult due to their low abundance and the inherent physical properties of lipids. We quantified PIPs extracted from cell lysates using direct detection on nitrocellulose membranes and in competitive ELISAs. Using specific antibodies and binding proteins we developed assays able to differentiate closely related phosphoinositides. These non radioactive assays employ synthetic di-C16 lipids as standards and are able to measure as little as 1-2 pmol of cellular PIPs. Human breast cancer cell lines MDA-MB-231 and MDA-MB-468 (PTEN deficient) were evaluated using activators and inhibitors of the PI3K pathway. We quantified PIPs from 231 and 468 cells using a biomass of several million cultured cells per assay point. We found 468 cells under hydrogen peroxide stress dramatically increased PtdIns(3,4,5)P3 and PtdIns(3,4)P2 levels while PIPn levels from 231 cells increased at a slower rate and at much lower concentrations. Levels of these two signaling PIPs were blocked by the PI3K inhibitor, wortmannin suggesting PI3K is directly involved. Lipid products of both class I and class II PI3Ks are increased by hydrogen peroxide stress in both PTEN normal and PTEN deficient breast cancer cell lines while pre-incubation with wortmannin attenuated both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 increases. Hyperactivation of class I and class II PI3K in the absence of PTEN regulation (468 cells) leads to excessive 3\#8217; phosphorylated phosphoinositide levels that are known to activate Akt, leading to uncontrolled cell growth and proliferation. We expect the ability to quantify individual phosphoinositides from cells will help assign specific biological functions to these important lipid second messengers. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5258.