Apoptosis in chronic lymphocytic leukaemia

B cell chronic lymphocytic leukaemia (B-CLL) is the most common adult leukaemia in the western world. The disease is characterised by the accumulation of a CD5+ B cell clone. Drug resistance is a major problem in B-CLL and complete remissions are uncommon. The lymphoaccumulative nature of B-CLL implies that dysregulation of the apoptotic process may be responsible for the development and progression of the disease. B-CLL cells were freshly isolated from patients, and an in vitro apoptosis sensitivity assay was developed using flow cytometric techniques. Initial studies confirmed the existence of 'spontaneous apoptosis' when B-CLL cells were cultured in vitro, and demonstrated a strong correlation between sensitivity to spontaneous apoptosis and sensitivity to apoptosis induced by the chemotherapeutic drug chlorambucil in vitro. Immunoblotting of chlorambucil and prednisolone treated B-CLL cells demonstrated the expression and activation of caspases -3, -7, and -8 in all samples analysed, whilst activation of caspase-2 was seen in cells from only one patient. Activation of caspases -3 and -7 was accompanied by the proteolysis of the DNA repair enzyme, poly (ADP-ribose) polymerase (PARP). Induction of apoptosis and activation of all the caspases was inhibited by the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.fmk). These results demonstrated a key role for the activation and processing of caspases in the execution phase of apoptosis in B-CLL cells. The B cell growth factors interleukin-4 and CD40 were demonstrated to strongly influence survival of B-CLL cells in in vitro culture, and also to modulate the response of the cells to chemotherapeutic drugs. Investigations into Fas induced apoptosis in B-CLL cells demonstrated the expression of the adapter protein, FDD, but no overexpression of the capase-8 inhibitory protein, c-FLIP. Additionally, B-CLL cells did not show rapid assembly of the death inducing signalling complex (DISC) in response to stimulation of Fas receptor, implying that these cells may preferentially utilise the Bcl-2-inhibitable Type II (mitochondrial) pathway of apoptosis induction, underlining the important role that Bcl-2 plays in determining the apoptotic sensitivity of B-CLL cells.