The role of pro-apoptotic BH3-only proteins in survival and differentiation of lymphocytes
Cell Biology
Final Report Abstract
In this project we addressed questions of the regulation of apoptosis by members of the Bcl-2-family of proteins. Due to the high number of such proteins, whose expression and interaction is basically impossible to control in an intact cell, much of their function is still unknown. Adding to this, ‘physiological’ apoptosis is not easy to study. Models amenable to cell biological and biochemical study are mostly tumour cell lines, which do not undergo spontaneous apoptosis. More physiological situations of apoptosis, such as death by growth factor limitation, are mostly restricted to small populations of primary cells that have to be isolated from animals or humans. We had established a model that to some extent can get around these problems. We used mouse haematopoietic progenitor cells (‘LMPP’), which can be expanded by conditional activation of the oncogene Hoxb8, but which are still sensitive to factor withdrawal and which can differentiate in vitro along several cellular lineages. We had to overcome some not entirely expected problems, like variation between cell lines and purification of cells during and upon differentiation. Eventually we settled on the analysis of LMPP (progenitors) and differentiation along the B lymphocyte lineage, where the cells reached a stage between pro- and pre-B cells. We analysed the role of individual Bcl-2- family members, focusing on cells lacking one or several of the ‘BH3-only’ proteins, the initiators of apoptosis within the Bcl-2-family. We confirmed some earlier results and made substantial new observations. Thus, the BH3-only protein Noxa was very important for apoptosis in LMPP but not in the differentiated cells; the reverse was the case for the protein Puma. Bim was the most important protein (this had been known), but in its absence the respective roles of Noxa, Puma and Bmf (Bmf was found not to play a role) could be assessed. We were able to measure the expression levels of the major Bcl-2-family proteins during B cell differentiation and noticed a number of striking differences. We further tested the susceptibility of the cells not only to the ‘physiological’ pro-apoptotic stimulus of factor withdrawal but also to the relatively recently introduced small molecule inhibitors of Bcl-2, Bcl-XL and Mcl-1. We further started analyzing complex composition within the Bcl-2-family by co-immunoprecipitation studies; these experiments are currently completed. The results first show the suitability of this model to the in-depth analysis of apoptosis regulation by proteins of the Bcl-2-family. They further identify some unexpected steps in this regulation during development, as well as unexpected interactions within the family. The sensitivity to Bcl-2-family inhibitors is probably of principal importance (with the limitations that we have only studied it here in mouse cells), because these inhibitors are more and more being used in the clinic. Lastly, the discovery of the importance of BH3-only proteins other than Bim in apoptosis regulation is, in our view, a significant contribution to the understanding of mitochondrial apoptosis.
