Constitutive Nuclear p65 NF-kB Expression Predicts for Spontaneous Apoptosis and In Vitro Sensitivity to Fludarabine in CLL Cells.

B-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B-cells in the blood, secondary lymphoid tissues, and bone marrow. The leukemia cells primarily are arrested in the G₀/G₁ phase of the cell cycle and appear resistant to programmed cell death. Despite their apparent longevity in vivo, CLL cells typically undergo spontaneous apoptosis in vitro although there is considerable heterogeneity in the timing and extent of this process. In this study we investigated the degree of this apoptosis in vitro, and its association with constitutive nuclear p65 NF-kB expression (n = 25). Apoptosis was measured by annexin V / propidium iodide labeling using flow cytometry and nuclear NF-kB expression was evaluated using electrophoretic mobility shift assay and quantified using a p65 ELISA. In contrast to previous reports, we found constitutive NF-kB activity varied in our patient cohort with a trend towards higher expression in samples with unmutated V_H genes, high CD38 expression and high ZAP-70 expression. Importantly, we demonstrated an inverse correlation between the percentage of Annexin V-positive cells and nuclear p65 NF-kB expression (r² = − 0.21; P = 0.02) suggesting that high levels of p65 NF-kB are cytoprotective in CLL cells. We then investigated the relationship between p65 NF-kB and in vitro response to fludarabine. Sensitivity to fludarabine was evaluated using LD₅₀ values calculated from sigmoidal dose-response curves. These LD₅₀ values positively correlated with nuclear p65 NF-kB activity (r² = 0.25; P = 0.006) indicating that in vitro drug resistance to fludarabine may be mediated, at least in part, through the transcriptional effects of NF-kB. Taken together, our data indicate that NF-kB is a key regulator of both spontaneous and drug induced apoptosis in CLL cells and is therefore a promising therapeutic target for the treatment of this incurable condition.