Lymphoid Microenvironment Inhibits Apoptosis in B-CLL Cells: Involvement of PI3-K/AKT Pathway and PTEN.

The activation of anti-apoptotic mechanisms in the leukemic B cells in chronic lymphocytic leukemia (B-CLL) through the interaction with their microenvironment may lead to prolonged survival and the accumulation of the malignant clone. The aim of this study is to elucidate the influence of the lymphoid microenvironment in the activation of the potent anti-apoptotic PI3-K/Akt pathway and to investigate the pattern of expression of the tumor suppressor PTEN in B-CLL. Stromal fibroblasts of bone marrow (BMF), spleen (SF) and lymph gland (LGF) were used as an in vitro model for lymphoid microenvironment. Pharmacological inhibitors and siRNAs against PI3-K and Akt were applied to explore the anti-apoptotic effect of this pathway in B-CLL. The results showed that co-cultivation of B-CLL cells with human BMF, LGF, and SF significantly inhibited apoptosis and prolonged survival of the leukemic cells in comparison to suspension cultures. To explore the involvement of PI3-K/Akt pathway in the anti-apoptotic effect of stromal fibroblasts, co-cultures were performed in presence of PI3-K inhibitors (wortmannin or LY294002) or siRNAs against PI3-K and Akt1. These inhibitors significantly reduced the supportive effect of stromal fibroblasts and induced apoptosis in B-CLL cells. The leukemic cells were more sensitive to PI3-K inhibition than T cells, monocytes and stromal fibroblasts. Induction of apoptosis was associated with a significant decrease in the intracellular levels of PIP3, PI3-K, PDK1, Akt1, NF-kappa B, IKK, and dephosphorylation (activation) of PTEN. Since PTEN activity, as a negative regulator for PI3-K signalling, is controlled by its phosphorylation at the tail domain, we studied the pattern of PTEN protein expression in B-CLL. Western blotting demonstrated that the total PTEN in PBMC of B-CLL patients (n=40) is comparable to healthy individuals (n=8). However, using phosphospecific anti-PTEN antibody demonstrated that samples of B-CLL patients highly express phosphorylated (inactive) forms of PTEN in comparison to healthy persons. In conclusion, the results demonstrate that PI3-K/Akt pathway is involved in inhibition of apoptosis of B-CLL cells and suggest that interaction of the leukemic cells with lymphoid microenvironment may lead to the activation of this pathway. The data also suggest that targeting this pathway represents a feasible therapeutic approach in B-CLL.