Altered Expression of PP2A Regulatory Subunits in Chronic Myelogenous Leukemia: Identifying Targets for Improved Therapies.

Abstract Over 90% of chronic myelogenous leukemia (CML) cases are associated with constitutive activation of the BCR/ABL oncoprotein, which induces transformation of hematopoietic stem cells. Inhibition of BCR/ABL with imatinib has shown remarkable success in treating CML patients. However, the emergence of resistance is becoming a major clinical problem. To direct the development of improved therapies, signaling pathways activated by BCR/ABL must be identified. The aim of this project is to determine whether the activity and expression of the tumor suppressor protein phosphatase 2A (PP2A) is altered in the presence of BCR/ABL. PP2A is a serine/threonine phosphatase that is composed of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B), of which there are three different families. Each family of PP2A-B subunits has a number of isoforms that determine the substrate specificity, subcellular localization and catalytic activity of the enzyme. In this study we show that transfection of BCR/ABL into FDC-P1, a mouse factor-dependent myeloid cell line, increased tyrosine phosphorylation on the PP2A-C subunit, and this correlated with a reduction in enzymatic activity. Imatinib treatment rescued PP2A activity in wild type BCR/ABL+ cells, but not the imatinib-resistant Y253F BCR/ABL+ cells. Importantly, pharmacological enhancement of PP2A by forskolin and FTY720 resulted in reduced proliferation in both cell lines. Furthermore, we have demonstrated for the first time that BCR/ABL increases the expression of three specific PP2A-B subunit isoforms - B55α, B56α, and B56δ. It is predicted that enhanced levels of these subunits cause displacement of alternate PP2A-B isoforms to alter enzyme composition and hence the specificity of PP2A. To determine the functional role of each individual B subunit, shRNA studies are being performed. These findings confirm that functional inactivation of PP2A is an essential component of BCR/ABL-induced leukemogenesis in CML, and provide novel insight into the mechanisms by which this may occur. Elucidating the importance of each regulatory subunit may identify novel targets to develop improved therapies for CML patients.</jats:p

High throughput analysis of endogenous glutamate release using a fluorescence plate reader

Recent discoveries of different modes of exocytosis and a plethora of molecules involved in neurotransmitter release has resulted in demand for more rapid and efficient methods for monitoring endogenous glutamate release from various tissue sources. In this article, we describe a high throughput microplate version of the enzyme-linked fluorescence detection method for the measurement of released glutamate, which utilises glutamate dehydrogenase, and the reduction of NADP to NADPH. Previous versions of this method rely upon cuvette-based fluorimeters for detection that are limited by large sample volumes and small numbers of samples that can be measured simultaneously. Comparison between the two methods shows that the microplate assay has comparable performance to the cuvette-based assay but has the capacity to analyse many times more samples in a given run. This increased capacity provides improved experimental design opportunities, higher experimental throughput and better comparison between experimental conditions. (c) 2005 Elsevier B.V. All rights reserved

Supplementary Figure Legends 1-10 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT&lt;sup&gt;+&lt;/sup&gt; Cancers

Supplementary Figure Legends 1-10 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT&lt;sup&gt;+&lt;/sup&gt; Cancers</jats:p

Supplementary Figure 3 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT&lt;sup&gt;+&lt;/sup&gt; Cancers

Supplementary Figure 3 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT&lt;sup&gt;+&lt;/sup&gt; Cancers</jats:p

Supplementary Figure 2 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT&lt;sup&gt;+&lt;/sup&gt; Cancers

Supplementary Figure 2 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT&lt;sup&gt;+&lt;/sup&gt; Cancers</jats:p

Supplementary Figure 2 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT&lt;sup&gt;+&lt;/sup&gt; Cancers

Supplementary Figure 2 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT&lt;sup&gt;+&lt;/sup&gt; Cancers</jats:p