Abstract
Chronic myeloid leukemia (CML) is driven by the constitutively activated Bcr-Abl tyrosine kinase, which causes deficiency in CXCR4-mediated migration of CML cells to bone marrow (BM) stroma. We have recently demonstrated that exposure of CML cells to imatinib under stromal co-cultures results in increased CXCR4 surface expression, enhanced migration of CML cells towards stromal cell layers and non-pharmacological resistance to imatinib (Jin, Mol Cancer Ther2008;7:48). Lipid rafts are plasma membrane microdomains, highly enriched in cholesterol, sphingolipids and in signaling molecules, which act as signal transduction platforms for a variety of intracellular processes. Lyn is a Src-family tyrosine kinase that is a downstream target of Bcr-Abl, and frequently localizes in lipid raft fractions. Binding to Bcr-Abl results in the constitutive activation of Lyn which impairs SDF-1 Ptasznik, J Exp Med2002;196:667). In this study, we investigated the effects of the tyrosine kinase inhibitor imatinib on the localization of Lyn in the lipid raft structures of CML cells under conditions mimicking the BM microenvironment. Imatinib treatment significantly increased cell surface CXCR4 expression levels in KBM5 CML cells only under mesenchymal stem cell (MSC) co-culture conditions as determined by FACS analysis (p<0.01). However, no significant difference in total CXCR4 protein levels was observed in control and imatinib/MSC co-cultured KBM5 cells by immunoblotting. These findings were confirmed by confocal microscopic analyses, whereby direct coculture of imatinib-treated KBM5 cells with MSC resulted in the increased expression of CXCR4 protein levels on the KBM5 cell surface without change in intracellular protein levels. In turn, KBM5 cells treated with imatinb in the absence of MSC, or co-cultured with MSC alone, showed no significant upregulation of surface CXCR4 expression. Analysis of lipid raft fractions using discontinuous sucrose density gradient fractionation demonstrated that Lyn strongly localized to lipid rafts in imatinib(+)/MSC(+) KBM5 cells compared to control KBM5 cells (5.2-fold increase in the ratio of Lyn to the raft marker flotillin-1). On the contrary, imatinib(+)/MSC(−) or imatinib(−)/MSC(+) conditioned KBM5 cells expressed similar levels of Lyn/flotillin in raft fractions. No significant difference in the levels of total or phosphorylated (Tyr396 and Tyr507) Lyn in whole cell lysates was detected by immunoblotting under all tested conditions.In conclusion, these findings demonstrate, for the first time, that Bcr-Abl oncoprotein inhibits Lyn trafficking to lipid raft microdomains in CML cells. Inhibition of Bcr-Abl by imatinib under stromal co-culture conditions promotes Lyn localization to the lipid rafts which in turn results in increased CXCR4 cell surface expression. These findings indicate that blockade of Lyn expression may ameliorate microenvironment-mediated resistance to tyrosine kinase inhibitors in CML.
Dynamics of putative raft-associated proteins at the cell surface
