Study of nuclear targets of phosphatidylinositol-3 kinase in lymphocytes.

Pathways regulated by Phosphotidylinositide-3-kinase (PI3K) have emerged as important mediators of cell proliferation and survival. When altered, several components of this pathway have been identified to contribute towards a wide range of human malignancies. PI3K has been implicated in the development of several EBV-associated malignancies of both lymphoid and epithelial origin. These include Burkitt's lymphoma, Hodgkin's disease and nasopharyngeal carcinoma. Although progress has been made in dissecting the pathways regulated by PI3K, the key components contributing to lymphocyte transformation have not been fully characterised. This study sought to investigate downstream targets of PI3K in lymphocytes in order to further our understanding of the contribution of PI3K signalling to lymphocyte proliferation and survival, particularly within the context of EBV-associated B-cell lymphomas. Initial work in this study revealed that a component of the mammalian ribosome, S6-ribosomal protein, is a major target for PI3K activation in transformed lymphocytes. In order to study PI3K and EBV regulated proteins on a larger scale, the technology of two-dimensional electrophoresis (2DE) was employed. The use of 2DE in combination with a PI3K inhibitor did not allow the identification of PI3K regulated proteins. However, three EBV regulated proteins were detected in the B-lymphocyte nucleus using this technology. The technology was further developed to study the post-translational modifications of DNA bound transcription factors. This detected multiple isoforms of the cAMP-response element binding protein (CREB), signal transducers and activators of transcription 1 (STAT1) and forkhead box O (FOXO) transcription factors in the nuclei of EBV immortalized lymphocytes. More detailed analysis of the PI3K regulated pro-apoptotic transcription factor, FOXO1, revealed that this protein is downregulated in EBV positive cells at both the transcriptional and translational levels. This downregulation was shown to directly correlate with the protein expression of a known target gene activated by FOXO1, Bcl-6, and to inversely correlate with protein levels of Cyclin D2, a target transcriptionally repressed by FOXO1. Further investigations into the mechanisms by which EBV downregulates FOXO1 implicated a role for two EBV encoded proteins, Latent membrane protein-1 (LMP1) and LMP2A in the downregulation of both FOXO1, and its target gene, Bcl-6. In conclusion, this work has explored the use of antibody detection and proteomic techniques for the identification and analysis of nuclear proteins and transcription factors regulated by PI3K and EBV. Together, these investigations have deepened our understanding of the molecular changes that occur in lymphocytes in response to EBV infection, and how EBV may influence malignancy