Diverse roles of the Bcl-2 family proteins in hemopoietic cell regulation

In this thesis, the roles of Bcl-2 family proteins in hemopoietic cell regulation were investigated. We first examined the effects of phosphatidylinositol 3-kinase (PI3K) dependent survival signalling pathways in cytokine dependent hemopoietic cells. Following cytokine withdrawal or PI3K inhibition, there was a loss of FOXO3A phosphorylation, resulting in increased expression of FasL and Fas at the cell surface. However, the Fas mediated signalling did not appear to be involved in apoptosis of cytokine dependent hemopoietic cells. These results support the belief that mitochondrial mediated signals through regulation of Bcl-2 family proteins may play the major role in hemopoietic cell apoptosis. Amongst the pro-survival Bcl-2 family members, Bcl-2 and Bcl-xL are assumed to have a redundant function. To explore the differential ability of Bcl-2 and Bcl-xL in protecting cells against apoptosis, we over-expressed these proteins in cytokine dependent hemopoietic cell line FDCP-1. Based on our results, Bcl-2 appears to be a more potent pro-survival protein than Bcl-xL against apoptosis induced by cytokine withdrawal. In addition to their localization at the mitochondria, Bcl-2 family members also localize at ER. To examine the physiological relevance of the membrane targeting of Bcl-xL, we used Rat-1 fibroblast cell lines over-expressing Bcl-xL mutants that were targeted to ER, mitochondrial outer membrane, or wild type Bcl-xL and showed that the ER targeted Bcl-xL was as effective or even more effective than the mitochondrial targeted or wild type Bcl-xL against certain cytotoxic stimuli. A number of studies have shown involvement of Bcl-2 family proteins in processes other than apoptosis. We explored a role of Mcl-1 in cell cycle regulation, DNA damage checkpoint response, and cellular differentiation and found an interaction between Mcl-1 and the cell cycle regulatory protein Cdk-1 in nuclear compartment. In addition, Mcl-1 was found to associate with the DNA damage checkpoint regulator, Chk-1, and the hallmark of DNA damage checkpoint response, phospho-histone H2AX. Mcl-1 level also increased in HL-60 cells upon induction of cellular differentiation by PMA. However, over-expression of Mcl-1 in these cells did not appear to enhance cellular differentiation. We, therefore, concluded that Mcl-1 might not play a prominent role in cellular differentiation.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat