Activation of AKT and Enhanced Resistance to Apoptosis Induced by Surface IgM Ligation in ZAP-70-Positive Chronic Lymphocytic Leukemia B Cells.

Signaling through the B-cell receptor (BCR) for antigen has been implicated to play a role in the pathogenesis and/or progression of chronic lymphocytic leukemia (CLL). Previously we found that BCR ligation on CLL cells that expressed ZAP-70 induced significantly higher levels of phosphorylation in p72Syk, BLNK, and phospholipase C-gamma than did BCR ligation on CLL cells that lacked expression of this protein tyrosine kinase. We hypothesized that CLL B cells that express ZAP-70 also may have enhanced levels of AKT activation following BCR ligation that is associated with increased resistance to apoptosis. CLL B cells were stimulated via surface IgM ligation and monitored at various time points for phosphorylation of cytoplasmic signaling molecules via immunoblot analyses and for viability by flow cytometry after staining with propidium iodide and DiOC6 to detect changes in mitochondrial membrane potential associated with apoptosis. We found that CLL cells that expressed ZAP-70 experienced significantly higher levels of AKT phosphorylation within 10 minutes following BCR-ligation than did ZAP-70-negative CLL cells. Over time a significantly higher proportion of ZAP-70-negative CLL cells were induced to undergo apoptosis by BCR-ligation than similarly treated CLL cells that were ZAP-70 positive. The differences between these two groups in leukemia cell viability over time after BCR ligation could be abrogated by addition of LY294002, a PI3-K inhibitor, to the CLL cell cultures 30 minutes prior to surface IgM ligation. We conclude that ZAP-70-positive CLL cells are relatively resistant to apoptosis induced by surface IgM ligation under the experimental conditions used, a characteristic that is associated with enhanced phosphorylation of AKT and activation of AKT-dependent pathways following BCR ligation. These studies support a model proposing that BCR ligation induces signaling that results in enhanced growth and/or survival of CLL cells that express ZAP-70 relative to that of leukemia cells that lack expression of this tyrosine kinase. Because expression of ZAP-70 typically is associated with expression of unmutated Ig V genes in CLL, the improved signaling afforded by expression of this tyrosine kinase may account in part for the greater tendency for disease progression observed in patients with CLL cells that use unmutated Ig V genes.