Medulloblastoma cells do not contain detectable amounts of the bZIP protein Zhangfei. However, previous work has shown that expression of this protein in cells of the ONS-76 line, derived from a human medulloblastoma, causes the cells to stop growing and develop processes that resemble neuritis (a characteristic of differentiated neurons). Zhangfei-expressing cells eventually die. My objective was to determine the molecular mechanisms by which Zhangfei influences ONS-76 cells. My strategy was to infect ONS-76 cells with adenovirus vectors expressing either Zhangfei or the control E. coli protein â-galactosidase (LacZ) and then to compare the following parameters in Zhangfei and LacZ-expressing cells: a) markers of apoptosis, autophagy and macropinocytosis (the three main pathways of cell death); b) transcripts for genes involved in neurogenesis and apoptosis; c) phosphorylation of peptide targets of selected cellular protein kinases; and d) active transcription factors. Zhangfei-expressing cells appeared to succumb to apoptosis as determined by the expression of phosphatidylserine on the cell surface and intensity of nuclear staining with the DNA dye Hoechst. Increased staining for autophagic vesicles and upregulated expression of autophagy response genes in these cells indicated that they were undergoing autophagy, possibly associated with apoptosis. My analysis of steady-state transcripts for genes involved in apoptosis and neurogenesis and functional protein kinases in Zhangfei-expressing cells indicated that the mitogen-activated protein kinase (MAPK) pathway was active in these cells. In addition, I found that the transcription factor Brn3a as well as factors implicated in differentiation were also active. These observations led me to hypothesize that Zhangfei enhances the expression of Brn3a, a known inducer of TrkA, the high-affinity receptor for nerve growth factor (NGF). TrkA then binds in an autocrine manner to NGF, triggering the MAPK pathway and leading to differentiation of ONS-76 cells into neuron and glia-like cells, eventually bringing about cell death by apoptosis and autophagy. I tested this hypothesis by showing that Zhangfei could enhance transcription from the isolated Brn3a promoter, that ONS-76 cells produce NGF as detected in a bioassay, and that antibodies against NGF and inhibitors of TrkA and selected components of the MAPK pathway could partially restore the growth of Zhangfei-expressing ONS-76 cells. My work supports previous work highlighting the importance of NGF-TrkA signaling in the outcome of medulloblastomas and shows how Zhangfei is able to trigger this pathway.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-06222011-145925 |
| Date | 11 July 2011 |
| Creators | Bodnarchuk, Timothy |
| Contributors | Mousseau, Darrell, Misra, Vikram, Napper, Scott, Bonham, Keith, Hill, Janet |
| Publisher | University of Saskatchewan |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-06222011-145925/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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