Analysis of B-cell receptor signal transduction in B-cell chronic lymphocytic leukemia by phosphospecific flow-cytometry

B-cell chronic lymphocytic leukemia (B-CLL) is a clonal lymphoproliferative disease characterized by the expansion of mature CD5+ B-cells. Recent studies indicated that variability in clinical outcomes observed in CLL patients is related to differences in the ability to transduce B-cell receptor (BCR)-mediate signals. The BCR signal transduction routes include, in the initial phases, Syk tyrosin kinase and BCR prolonged stimulation has been associated to activation of kinases including mitogen activated protein kinases (MAPK). In this study we used phosphospecific flow cytometry to study single-cell signaling triggered by BCR in peripheral blood B-cells from 21 B-CLL patients. The following proteins were analyzed: Syk, MAPK (Erk, p38, JNK) and NF-kappaB, either in basal condition or after BCR cross-linking achieved by treatment with anti-IgM F(ab’)2. Most cases showed constitutive phosphorylation of Syk and NF-kappaB (86% and 93%, respectively). Erk was constitutively phosphorylated in 81% cases, JNK in 5%, and p38 in 0% (Table 1). The BCR cross-linking increased the constitutive phosphorylation of Syk in 50% cases, of Erk in 27%, and of NF-kappaB in 21%. No cases showed p38 or JNK increased phosphorylation (Table 1). Recent works shows that efficient activation of BCR signaling depends on inactivation of phosphatase proteins (PTPs), and that endogenously generated H2O2 is an important transiently PTPs inhibitor. Therefore, we cross-linked the BCR in the presence of H2O2. In this condition we observed an increase of phosphorylation of all proteins with respect to treatment with anti-IgM alone (Table 1). Furthermore, the treatment of B-CLL cells with H2O2 alone induced increased phosphorylation of signaling proteins with respect to the constitutive level (Syk in 21%, p38 in 56%, Erk in 38%, JNK in 9%, and NF-kappaB in 43% cases, Table1). Conversely, PTP inhibition in normal B cells did not significantly modify the phosphorylation of these proteins. Taken together, these results revealed a clear-cut remodeling of BCR-mediated signaling in B-CLL cells with respect to normal B cells, thus confirming and extending previous results obtained by Western blot analysis. Furthermore, this study provides the first analysis of BCR signaling by phosphospecific flow cytometry in B-CLL cells and indicate that single-cells measurement of phosphoproteins in response to BCR engagement enables to define cell network phenotypes in B-CLL by multidimensional molecular profiles of signaling.