Dishevelled Proteins of Wnt Signaling Pathways Are Highly Expressed in Chronic Lymphocytic Leukemia

Abstract 4583

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world and is characterized by progressive accumulation of CD5+, CD19+, and CD23+ B cells in the blood, bone marrow, and secondary lymphoid organs. The progressive accumulation of malignant B cells is partly due to defective apoptosis. An important constitutively activated signaling pathway in CLL cells might be the Wnt signaling cascades that include the Wnt/b-catenin pathway and the non-canonical Wnt pathway. These pathways might be activated downstream of the constitutively phosphorylated ROR1. Dishevelled family proteins (Dvl1, Dvl2, and Dvl3) are important cytoplasmic mediators of Wnt signaling and have recently been shown to be expressed in many cancer types. The expression and precise roles of individual Dvl proteins are however not clear.

The aim of the study was to characterize the expression of Dvl1, Dvl2 and Dvl3 proteins in progressive and non-progressive CLL and compare to normal white blood cells (PBMC) and to assess the expression of other important proteins of the canonical pathway as b-Catenin and GSK-3β as well as the non-canonical pathways as GSK-3α, PKC, ERK and AKT respectively.

Leukemic cells from progressive (n=9) and non-progressive (n=9) CLL patients were isolated by Ficoll gradient centrifugation as well as PBMC from healthy donors (n=9). Six cell lines including four CLL cell lines EHEH, CII, I83, 232 B4, and the Jurkat and Lucas cell lines were also used. Dvl 1, Dvl 2, Dvl 3 proteins were analyzed by Western blot and densitometric calculations as well as PKC, GSK-3α, AKT and ERK using antibodies against the total protein and the phosphorylated protein respectively. Immunoprecipitation was performed to check the phosphorylation status of Dvl proteins.

Overexpression of Dvl1, Dvl2, and Dvl3 was detected in all progressive and non-progressive CLL patients and the six cell lines. There was a significantly higher expression of Dvl1 and Dvl3 in progressive compared to non-progressive CLL patients (p<0.01 and p<0.01 respectively). In contrast to Dvl1 and Dvl3, Dvl2 was highly expressed in non-progressive as compared to progressive CLL patients. Dvl1 was phosphorylated at serine residues while Dvl2 was phosphorylated at tyrosine residues. b-Catenin followed the same expression pattern as Dvl2 being highly expressed in non-progressive CLL as compared to progressive CLL patients (p<0.001) whereas GSK-3β phosphorylation was significantly higher in progressive than non-progressive CLL patients (p<0.01). There was no significant difference in the degree phosphorylation of PKC and GSK-3α comparing non-progressive CLL, progressive CLL and healthy donors.

In summary Dvl1, Dvl2 and Dvl3 are highly upregulated in CLL patients and CLL cell lines but the expression in normal PBMC is almost negligible. Dvl1, Dvl2 and Dvl3 may have a key role in the transduction of Wnt mediated signals in CLL.