Protein Kinase C-Epsilon Is An Important Component of the Lyn Kinase Signaling Pathway in the Apoptotic Resistance of B- Chronic Lymphocytic Leukemia Cells.

Abstract

Although several mechanisms of apoptotic defects have been implicated in B- chronic lymphocytic leukemia (CLL) cells, the exact signaling pathways underlying these defects remain unresolved. Lyn kinase and protein kinase C (PKC)-epsilon play a critical role in B-cell antigen receptor signaling. They are also known to be negative regulators of apoptosis, and are linked to chemo-resistance. Because activation of PKCs results in their translocation from the cytosol to distinct subcellular locations in which they perform their specific functions, we examined the role of Lyn kinase in the cellular localization of PKC-epsilon in B-CLL cells and its link to the apoptotic defects. Untouched peripheral blood B-CLL cells or nonmalignant B cells were isolated from PBMCs of B-CLL patients or normal blood donors. Cytoplasmic and nuclear fractions of B-CLL cells and nonmalignant cells were prepared and examined for PKC-epsilon localization by Western blot analysis. Unlike the cytoplasmic localization in nonmalignant cells, a large fraction of PKC-epsilon in B-CLL cells was located in the nuclear fraction. Nuclear localization of PKC-epsilon in B-CLL cells was linked to the transcriptional activation of the antiapoptotic genes such as Mcl-1, XIAP, and Bcl-2, as well as NF-kappaB activation and VEGF production., both strongly implicated in the apoptotic resistance of B-CLL cells. Treatment of B-CLL cells for 16-20 hours with 10 micromolar Lyn-specific inhibitor peptide targeting a unique interaction site within Lyn (Cancer Res. 2004; 64:1058) resulted in redistribution of PKC-epsilon from the nuclear to the cytoplasmic fraction, decreased expression of the antiapoptotic genes as much as 60%, and decreased the viability of the leukemic cells. In addition, the Lyn-specific inhibitor peptide induced more than 50% inhibition of NF-kappaB p65 nuclear translocation and VEGF production in B-CLL cells but not in nonmalignant B cells. Similar results were obtained when a selective inhibitor peptide of PKC-epsilon translocation but not the negative control peptide was used. Our results suggest that PKC-epsilon is a downstream target of Lyn kinase, and that inhibition of PKC-epsilon nuclear translocation may have therapeutic potential by targeting Lyn kinase signaling pathway for the treatment of B-CLL.