Investigation of the localization of the second messenger phosphatidylinositol-(3,4,5)-trisphosphate in chronic myeloid leukemia

Xenaki, D and Gray, A and Downes, C and Owen-Lynch, P (2002) Investigation of the localization of the second messenger phosphatidylinositol-(3,4,5)-trisphosphate in chronic myeloid leukemia. Experimental Hematology, 30 (6 Supp). p. 79. ISSN 0301-472X

Abstract

Phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) is a phospholipid second messenger generated by the activity of phosphatidylinositol 3-kinase in response to many hematopoietic growth factors. This activity is thought to be associated with the downstream survival and proliferative effects of such growth factors. PI3-kinase has also been shown to be activated in response to Bcr/Abl, the protein tyrosine kinase that is the hallmark of chronic myeloid leukemia. In order to confirm PI3-kinase activation it is necessary to prove that the levels of its main product PIP3 rise in vivo. The aim of this work is to assess changes in PIP3 in vivo in normal and Bcr/Abl-expressing hematopoietic progenitor cells and thus determine the role of this vital signal transduction pathway in normal hematopoiesis and leukemogenesis. We have generated a purified, baculovirus expressed, fluorescent probe for the detection of PIP3. The probe consists of a PH domain derived from GRP-1, a protein that specifically binds PIP3, fused to GFP. We have established a protocol for the use of this novel probe in fixed cell preparations and have verified its specificity for PIP3 by using binding competitors, such as inositol-(1,3,4,5)-tetrakisphosphate, and inhibitors of PIP3 generation, such as the specific inhibitor of PI3-kinase activity LY294002. Stimulation of normal hematopoietic cells with Interleukin-3 (IL-3) produces a consistent increase in the total amount of cellular fluorescence compared with unstimulated cells. In agreement with results obtained from in vitro PI3- kinase and PKB activity assays, Bcr/Abl activation also induces increased PIP3 generation in the absence of IL-3. Analysis of fixed cells stained with the GFPprobe by confocal microscopy confirms that IL-3 and Bcr/Abl both stimulate an increase in PIP3 levels. It also demonstrates that PIP3 localises in the plasma membrane and cytoplasm of resting cells, but it is generated within the nucleus of the cell after stimulation. These results thus present the exciting hypothesis that hematopoietic growth factors and Bcr/Abl can stimulate nuclear PI3-kinase activity to generate concentrated localized increases in this important second messenger.