Activation by CD40 Ligand Shifts Bcl-2 Family Protein Expression from Death-by-Default to Survival-by-Default.

Abstract

CLL cells survive and accumulate in vivo and yet leukaemic cells isolated from the peripheral blood rapidly die. We have used mouse fibroblast L-cells transfected with CD40 ligand (CD40L) in combination with IL-4 to reproduce some features of the in vivo microenvironment, which is essential for CLL cell proliferation. As anticipated we found that leukaemic cells survive and proliferate in this system. Also CD23 expression increases, but unexpectedly CD38 expression increased to varying degrees in all patients and CD5 expression decreased. Changes in CD23 and CD38 have been reported in CLL cells in lymph nodes and support the concept that we are able to some extent reproduce the in vivo microenvironment. Next we carried out a systematic analysis of Bcl-2 family proteins by Western Blot analysis. On tissue culture plastic and non-transfected L-cells Bcl-2 is the predominant pro-survival protein. The activator BH3-only protein Bim is expressed together with the sensitiser BH3-only Puma. Both pro-apoptotic Bak and Bax are expressed. By contrast on CD40L-L cell with IL-4 culture Bcl-2 expression decreases and the predominant prosurvival protein is Bcl-xL with a contribution from Mcl-1. The activator BH3-only protein is Bid rather than Bim and the sensitiser BH3 only proteins Puma, Noxa and Bad are expressed. Levels of Bak are significantly decreased but Bax expression is unchanged. Overall it appears that there is an excess of pro-survival Bcl-xL on CD40L-L cells/IL-4 with decreased amounts of Pro-apoptotic Bak. These findings are important when considering the place of treatments directed at pro-survival Bcl-2 family proteins. Large amounts of Bcl-xL is a reservoir to soak up the reduced amount of Bak suggesting that whilst Bcl-2 antagonists will tip the balance towards apoptosis in CLL cells activated by CD40L/IL-4 they are unlikely to be sufficient to cause cell death. However, leukaemic cells in the peripheral blood are dependent on Bcl-2 and have increased amounts of Bak. In this situation reduction in Bcl-2 will release sufficient Bak to cause apoptosis.