Roles of the phosphatidylinositol 3-kinase regulatory subunit p55gamma on regulating survival signaling in breast cancer cells
Thesis (Ph.D.)--Georgetown University, 2009.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. Phosphatidylinositol 3-kinase (PI3K), specifically its catalytic subunit, is involved in tumorigenesis. However, the roles of the regulatory subunit of PI3K in cancer are largely unknown. Our aim is to determine the roles of the PI3K regulatory subunit p55gamma on regulating the survival signaling of breast cancer cells.; Silencing of p55gamma significantly reduces cell growth and induces apoptotic cell death. As a result, breast cancer cells are less capable of forming colonies in vitro. Morphologically, p55gamma reduction induces multipolar mitosis, as indicated by immunostaining of alpha-tubulin. Silencing of p55gamma also causes cell cycle arrest in G2/M phase, and the G2/M checkpoint proteins are correspondingly modulated after p55gamma reduction. The up-regulation of cyclin B1 and phosphorylated Cdc2, as well as down-regulation of Chk-1 protein, confirm an increased proportion of cells lingering in mitosis phase and indicates that these cells might die from prolonged mitosis phase retention. Moreover, silencing of p55gamma results in down-regulation of the survival signaling protein-survivin, further supporting our hypothesis that p55gamma is involved in survival signaling. These data indicate that expression of p55gamma is necessary for cell growth; the silencing of p55gamma results in significant M phase retention, increased apoptosis, mitotic defects, and profound cell death in some cells. Interestingly, the survival signaling mediated by p55gamma might involve not only the conventional but also the non-conventional PI3K/Akt pathway, because not all cells with p55gamma silencing show reduced activation of Akt.; Upon activation of PI3K through epidermal growth factor stimulation, the localization of p55gamma, which is shown by a GFP-p55gamma fusion protein, shifted from the cytoplasm to not only the cellular membrane, but also the nuclear region. This suggests that a novel role of p55gamma might exert by interaction with the nuclear proteins. Furthermore, we demonstrate the formation of a protein complex consisting of calmodulin, p55gamma and Rb, and we show the change in the relative binding in the presence of calcium. These observations indicate that p55gamma may regulate cell signaling through interaction with these proteins.; In conclusion, our study has revealed for the first time that p55gamma plays a significant role in the survival signaling of breast cancer cells.
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