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THE DISCOVERY OF A NOVEL 105KDA COMPLEX COMPRISED OF
BUB3, C-JUN AND SUMO-1 AND ITS RELATIONSHIP TO
OVARIAN CANCER DEVELOPMENT
by
Jennifer Yeh Lee
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)
December 2009
Copyright 2009 Jennifer Yeh Lee
Object Description
| Title | The discovery of a novel 105kDa complex comprised of BUB3, C-JUN and SUMO-1 and its relationship to ovarian cancer development |
| Author | Lee, Jennifer Yeh |
| Author email | jenny.yeh@gmail.com; jensyeh@hotmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Pathobiology |
| School | Keck School of Medicine |
| Date defended/completed | 2009-10-09 |
| Date submitted | 2009 |
| Restricted until | Unrestricted |
| Date published | 2009-11-03 |
| Advisor (committee chair) | Dubeau, Louis |
| Advisor (committee member) |
Schonthal, Axel Widelitz, Randall B. Ladner, Robert D. |
| Abstract | This work is based on the hypothesis that post-translational modifications catalyzed by ubiquitin and SUMO play an important role in tumor development and progression. Nuclear protein extracts were obtained from a cell strain transfected with SV40 large T antigen derived from a benign epithelial ovarian tumor (ovarian cystadenoma) and from a subclone of the same strain that had undergone spontaneous immortalization in vitro. Both nuclear extracts were analyzed by two-dimensional gel electrophoresis followed by western blotting with antibodies against ubiquitin and SUMO-1. Two protein complexes, respectively 40kDa and 105kDa, appeared to be differentially ubiquitinated and sumoylated. Protein sequencing analyses revealed that both complexes contained BUB3, a checkpoint protein involved in mitotic regulation. Immunoprecipitation and western blot analysis along with 35S-methionine labeling experiments, GST-pull down experiments, and gel filtration experiments confirmed the findings of a novel sumoylated 105kDa complex containing BUB3, SUMO-1 and C-JUN. Formation of the 105kDa complex is likely to be via covalent bonds. It is upregulated in cells approaching crisis, which is characterized by a mitotic arrest, and remains elevated in cells that have spontaneously recovered from such arrest, which are typically near tetraploid. Immortal ovarian carcinoma cell lines that are near tetraploid, including ML9A, SKOV3, and CAOV3, show detectable amounts of this complex, while cell lines that are near diploid, such as HEY and HOC-7, do not. Treatment with siRNA against BUB3 leads to a decrease in the 105kDa complex as well as native BUB3. Treatment of the cells with paclitaxel, a microtubule inhibitor, leads to intracellular accumulation of the complex, while treatment with aphidicolin, an S phase inhibitor, leads to its decrease, suggesting that formation of this complex is cell cycle dependent, occurring later than the S phase and preceding microtubule assembly. |
| Keyword | BUB3; C-JUN; SUMO-1; mitotic checkpoint; aneuploidy; tetraploidy |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m2705 |
| Contributing entity | University of Southern California |
| Rights | Lee, Jennifer Yeh |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | cisadmin@dots.usc.edu |
| Filename | etd-Lee-3352 |
| Archival file | uscthesesreloadpub_Volume40/etd-Lee-3352.pdf |
Description
| Title | Page 1 |
| Contributing entity | University of Southern California |
| Repository email | cisadmin@dots.usc.edu |
| Full text | THE DISCOVERY OF A NOVEL 105KDA COMPLEX COMPRISED OF BUB3, C-JUN AND SUMO-1 AND ITS RELATIONSHIP TO OVARIAN CANCER DEVELOPMENT by Jennifer Yeh Lee A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PATHOBIOLOGY) December 2009 Copyright 2009 Jennifer Yeh Lee |
