Expression of ZAP-70 in B Cell Chronic Lymphocytic Leukemia Is Associated with a Greater Capacity To Activate NFκB Following Ligation of Surface Immunoglobulin.

Abstract

The NFκB transcription factors p50/p65 regulate the expression of genes encoding various growth-promoting factors and anti-apoptotic proteins, such as the cellular inhibitors of apoptosis (c-IAPs), Caspase-8/Flice-inhibitory protein (FLIP), A1 (also known as Bfl1), tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) and 2 (TRAF2). Furthermore, constitutive activation of NFκB has been observed in many tumor types, supporting the notion that activation of NFκB can play a causal role in tumor development and/or progression. Studies have shown chronic lymphocytic leukemia (CLL) cells experience activation of NFκB in vitro upon ligation of their surface immunoglobulin (Ig), which commonly possesses polyreactive-binding activity for many self-antigens. Other studies also have found that CLL cells from different patients vary in their capacity to undergo B-cell-receptor signaling following ligation of their surface Ig receptors, a capacity that appears associated with leukemia-B-cell expression of the zeta-associated protein of 70 kD (ZAP-70). We examined whether CLL B cell expression of ZAP-70 also was associated with the capacity to activate NFκB upon surface Ig ligation. For this we used CLL B cells of 8 different patients that expressed ZAP-70 and CLL B cells from 8 other patients that had negligible expression of this tyrosine kinase (as assessed by immunoblot and flow cytometric analysis). The CLL B cells of these two groups of patients had similar expression levels of surface, allowing us to use a F(ab')2 anti-human IgM (anti-μ) to effect comparable surface Ig receptor ligation. Following treatment with anti-μ, we observed early and sustained degradation of IκB-α, thereby releasing cytoplasmic p50/p65 to the nucleus - the hallmark of NFκB activation. Moreover, this was associated with subsequent increased expression of NFκB target genes. In contrast, similar events were not observed following treatment with anti-μ in the cases lacking expression of ZAP-70. Also, activation of NFκB in ZAP-70+ cases was associated with a greater release of intracellular calcium and calcium flux following treatment with anti-μ than observed in ZAP-70-negative cases. Both calcium flux and activation of NFκB induced by anti-μ in these leukemia cells could be inhibited by Cyclosporine-A, indicating that these responses were mediated via a calmodulin-calcineurin-dependent pathway. These studies reveal that expression of ZAP-70 in B cell CLL is associated with a greater capacity to induce activation of NFκB following ligation of surface Ig, a characteristic that might account for the more aggressive clinical behavior of patients with leukemia B cells that express this tyrosine kinase. Moreover, if constitutive activation via ligation of surface Ig with self-antigen in vivo leads to activation of NFκB, then targeting the calmodulin-calcineurin-dependent pathway might have therapeutic potential for this subset of patients with this disease.