Autologous plasma activates Akt/protein kinase B and enhances basal survival and resistance to DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia cells

We have studied the actions of autologous plasma on both basal and DNA dama ge-induced apoptosis in B-chronic lymphocytic leukaemia (B-CLL) cells. Apop tosis was quantified using morphological criteria and Western blot analysis for the apoptosis-specific p85 fragment of poly(ADP ribose) polymerase. Ce ll viability was estimated using the methyl thiazol tetrazolium bromide dye reduction assay. Plasma cultures showed lower rates of basal apoptosis as well as a decreased cytotoxic response to chlorambucil and gamma -radiation compared with cultures in fetal calf serum. Experiments using neutralizing antibodies suggested that the protective actions of plasma could not be ac counted for by interleukin 4, the interferons alpha or gamma or stromal cel l-derived factor 1, each of which have been shown to protect B-CLL cells fr om apoptosis in vitro. Plasma addition to B-CLL cells resulted in rapid act ivation of the Akt protein kinase, a key signalling enzyme that has been im plicated in anti-apoptotic signalling. LY294002, an inhibitor of phosphatid ylinositol 3'-kinase, blocked Akt activation by plasma. To the best of our knowledge, this is the first report to show that factors present in plasma promote basal survival of B-CLL cells and resistance to cytotoxic drugs via stimulation of the Akt cytoprotective-signalling pathway. Pharmacological blockade of this pathway may have potential in the development of novel the rapeutic strategies for B-CLL treatment.