Influence of ZAP-70 Expression On Migration of Chronic Lymphocytic Leukemia (CLL) and Lymphoma Cells.

Abstract 2345

Poster Board II-322

ZAP-70 (ξ-associated protein) is a tyrosine kinase (PTK) of the Syk/ZAP family normally expressed in T and NK cells. Increased ZAP-70 expression in CLL correlates with unmutated IgVH genes, a short time of progression, and a short survival. Mechanisms by which increased ZAP-70 protein expression can influence clinical outcome are not fully understood. After B-cell receptor (BCR) stimulation, activated ZAP-70 can cooperate with Syk, the tyrosine kinase of the BCR signal pathway normally expressed in B-cells, thus inducing activation of the CLL cells. Stimulation of the BCR leads to the triggering of Syk protein kinase to the cytoplasmatic tails of the receptor initiating subsequent activation of critical effector enzymes such as PI3K and PLCγ2. The PI3K/Akt pathway is related to survival and protection from BCR-induced apoptosi. Against this background, we analyzed the functional consequences of abnormal expression of ZAP-70 protein in B-lymphoma cell lines that normally does not express ZAP-70, particularly analyzing the impact of increased ZAP-70 expression oncell metabolism and cell migration. Mec-1 (CLL), Raji and Ramos (Burkitt) cell lines were stably transfected with the ZAP-70 expression vector pEGFP-ZAP-70 as well as the control pEGFP. BCR stimulation was induced in the stable Ramos cell line by IgM at several time points, and phosphorylation of downstream proteins was analyzed by Western Blot. Proliferation, cell cycle, calcium flux, adhesion molecules and cell migration were analyzed in all stable cell lines after ZAP-70 phosphorylation. After IgM stimulation, in Ramos cell line phosphorylation of ZAP-70 was observed at residue Y319 and, simultaneously, phospho-Syk expression was reduced. In addition, Erk protein was strongly activated in ZAP-70 transfected cell line compared to the control cell line, this activation lasting more than 24 hours. Notably, calcium flux detected by flow cytometry was higher in ZAP-70 transfected Ramos. Migration experiments showed that after IgM stimulation, transfected ZAP-70 Ramos migrated significantly more rapidly than cells transfected with the vector alone. Of note, after IgM stimulation, ZAP-70 transfected Ramos cells expressed a higher number of adhesion molecules (CXCR7 and others) on surface membrane than non-transfected cells. This effect on migration stimulation was also observed with the Raji cell line transfected with ZAP-70. In conclusion, addition of ZAP-70 expression to a heterologous B-cell system enhances BCR downstream signalling, calcium mobilization, and migration. Ongoing studies are determining the influence on the gene expression profile dependent of ZAP-70 expression. Altogheter, these data suggest that additional ZAP-70 expression in CLL and other lymphoproliferative disorders enhances tumoral activity, this explaining in part the poor clinical prognosis of patients with increased expression of ZAP-70.