Class IA phosphoinositide 3-kinases (PI(3)Ks) are critical control elements of signalling cascades with influence over cell survival, proliferation and migration. Mammals have 3 distinct class IA PI(3)K catalytic subunits. One of these, pi 106, is preferentially expressed in leukocytes. Previous murine genetic studies using strategies which target regulatory elements thereby disrupting class IA PI(3)K signalling indiscriminately of the catalytic subunits have established that PI(3)K are important for Kit signalling and mast cell homeostasis but not for mast cell activation and the allergic response. We have utilised a mouse model in which pi 108 is inactivated by introduction of a germline mutation in the ATP binding site, creating pi 105D910A , mimicking the effects of a pharmacological inhibitor for pi 105. Bone marrow mast cells (BMMCs) from pi 1 o D910A/D910A mice have severe defects in SCF/Kit responses including proliferation, migration and adhesion additionally BMMCs derived from these mice have a substantial defect in antigen receptor signalling and mast cell degranulation. pi 108D910A mice have tissue site-selective loss of mast cell populations and a substantial reduction in IgE/Ag-induced passive cutaneous anaphylaxis response. Using pi 108 isoform-selective inhibitors it is possible to replicate our genetic strategy and completely block the in vivo allergic immune response. Furthermore using such compounds and human mast cells we show that our findings have direct relevance to the human system.

Apoptosis is known for its physiological role in development, sculpting of tissues, and regulation of immune cells. Although much of the signalling pathway has been dissected, the mechanism of many apoptotic events remains undefined. Furthermore, the lack of knowledge of how cells commit to apoptosis has impeded our understanding of how this process is altered in disease states. This study aimed to determine the sequence of key apoptotic events using novel combinations of flow cytometric assays, and to investigate the irrevocable point-of-no-return. To induce apoptosis, Jurkat T-cells were treated with camptothecin, anti-CD95- antibody or cycloheximide, in the presence/absence of inhibitors. Various apoptotic events were detected by flow cytometry. Secondly, cells were treated with apoptotic inducers in the presence of caspase inhibitor, and the differential gene expression pattern was studied using microarray. Four hours after treatment with stimuli, apoptosis were detected in approximately 50% of the cells. This thesis presents evidence that cell cycle may play a decisive role. Transcriptional regulation of p21 and GADD45B were found to control cell-cycle arrest and cell death. Multicolour flow cytometric assays also demonstrated that a proportion of cells in the AnnV7AAD- population (conventionally defined as live cells) underwent mitochondrial depolarisation and caspase activation after camptothecin or anti-CD95 treatment.

The Bcl-2 family of proteins are critical regulators of mitochondrial function and deregulation during apoptosis. To this effect, the 'BH3-only' members of this family play an important role in initiating many of the apoptotic signalling pathways. This thesis focuses on the BH3-only protein Bid. We have used the active form of Bid, tBid, as bait in a yeast two-hybrid assay to identify novel interaction partners for this protein. In combination with a GST pull-down assay we identified two novel interaction partners for tBid. The proteins identified were Mcl-1 and EID-1. Mcl-1 is an anti-apoptotic Bcl-2 family member, although its role in apoptosis has not been as extensively studied as other anti-apoptotic proteins such as Bcl-2 and Bcl-XL. EID-1 was first identified as a pRb interacting protein that has been shown to inhibit differentiation of myoblasts. However, there is no data to suggest that EID-1 may have a role in apoptosis, and to date all data suggests that the protein has a nuclear function rather than a cytoplasmic one. During the course of this work, we observed that Mcl-1 is cleaved during apoptosis of Jurkat T cells. We have characterised this cleavage and found it to be as a result of caspase cleavage. There are two caspase cleavage sites in Mcl-1. These occur at Asp127 and Asp157. Many Bcl-2 family members are cleaved by caspases during apoptosis. In particular, anti-apoptotic family members cleaved by caspases are converted into pro-apoptotic proteins. In contrast to this we did not observe any pro- apoptotic activity associated with the cleavage fragments. We also found that the PEST region in Mcl-1 did not regulate its half-life in FDCP-1 cells, confirming results already observed in U937 cells. While further work needs to be carried out, the results presented here provide a number of important observations that may help to further the understanding of how both Bid and Mcl-1 contribute to apoptosis.

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Neuroblastomas are common paediatric tumours derived from the sympathoadrenal lineage. Neuroblastoma cells may arise from neuroblasts, which failed to differentiate or which were not eliminated by apoptosis at an appropriate stage of development. The aim of this thesis was to identify the signalling pathway by which cis-diamminedichloroplatinum (II) (CDDP) a chemotherapeutic agent, triggers caspase activation and apoptosis in the SH-SY5Y neuroblastoma cell line. An understanding of this may prove to be useful for developing better therapeutic agents for treating neuroblastoma and for understanding mechanisms of drug resistance. CDDP was found to induce apoptosis in SH-SY5Y cells via the mitochondrial death pathway, with cytochrome c release and activation of caspases-9 and -3. CDDP, a DNA damaging agent, activates p53 in SH-SY5Y cells and p53 is known to induce apoptosis via the mitochondrial pathway. Bcl-2 family members play a central role in the regulation of the mitochondrial death pathway and may have pro- or antiapoptotic activity. The pattern of expression of members of the Bcl-2 family following CDDP treatment was investigated to determine their regulatory role. PUMA (p53 upregulated modulator of apoptosis), a proapoptotic BH3-only protein and a direct transcriptional target of p53, was found to be upregulated at both the mRNA and protein levels during CDDP-induced apoptosis of SH-SY5Y cells. PUMA has three transcripts that encode PUMA- a, P and 5. PUMA-a and PUMA-(3 are proapoptotic and contain the BH3 domain. PUMA-a was identified as the transcript that increased during CDDP treatment in SH-SY5Y cells. Overexpression of PUMA-a in SH-SY5Y cells was sufficient to induce apoptosis. To identify other genes regulated by CDDP, we performed oligonucleotide microarray analysis using Affymetrix human genome-U133A microarrays and RNA extracted from SH-SY5Y cells treated with DMSO, transplatinum (an isomer of CDDP which does not induce cell death) and CDDP. The results provide a detailed picture of the changes in gene expression that occur following CDDP treatment.

Fas (CD95) is a member of the death receptor superfamily of proteins that are involved in the initiation of apoptosis as induced by the binding of extracellular ligands. At present, little is known about the precise mechanism by which the signal initiated by the interaction between Fas ligand (FasL) and Fas is transduced across the cell membrane to start the apoptotic signalling cascade. The first step in this pathway is the recruitment of the Fas-associated death domain protein (FADD) to the cytoplasmic death domain of Fas via a homotypic protein/protein interaction. This binding event occurs after receptor ligation apparently without any post translational modification such as phosphorylation. In order to better understand this event we have investigated the interaction between the death domains of the human Fas and FADD proteins both in vitro and in a cellular context. The reported interaction between the Fas and FADD death domains (Fas-DD and FADD-DD) was recapitulated using the yeast 2-hybrid assay. Recombinant proteins were then produced for NMR spectroscopy experiments. FADD-DD is highly expressed, and easy to isolate soluble at physiological pH. This domain is readily expressed as a histidine tagged domain. Fas death domain expresses at low levels and produces soluble aggregates when concentrated at a pH above 4. However, it was found that using a Gbl fusion protein to express Fas-DD overcomes these problems and allowed the production of Fas-DD for NMR experiments. NMR titration experiments showed that when these two proteins interact a large, soluble complex is formed. This may be significant in relation to the increasing evidence for the importance of Fas-receptor clustering in its signalling. Mutational analysis of the Fas death domain was also carried out. Here, various previously described as well as several novel point mutations were made on the surface of the Fas death domain to investigate their effect on FADD-DD binding. These mutations were assayed using yeast 2-hybrid methods, NMR analysis and in a cell based assay. In the cell based assay wild type and mutant Fas receptors were overexpressed in a human cell line with no endogenous surface Fas expression. These cells were then assayed for their ability to undergo FasL-induced apoptosis. It was found that residues from many surface regions of Fas-DD are crucial for the FADD-DD interaction. This observation has potentially important implications for the nature of the organisation of the death domains in the death inducing signalling complex (DISC) formed at the Fas receptor.

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