SV40 large T antigen (TAg) is a dominant acting oncoprotein that elicits transformation of many cell types and induces tumors in rodents. TAg induces transformation, in part, by disabling the functions of tumor suppressors such as pRb and p53. This dissertation is aimed to determine if inactivation of Rb and p53 are the major TAg activities required for transformation or if additional activities contribute.
To determine whether Rb-family protein inactivation by the J domain of TAg is required for induction of intestinal hyperplasia, we have generated transgenic mice that express a J domain mutant (D44N) in villus enterocytes. In contrast to wild-type T antigen, the D44N mutant is unable to induce enterocyte proliferation. Unlike mice expressing wild-type TAg, mice expressing D44N do not reduce the protein levels of p130 and are also unable to dissociate p130-E2F DNA binding complexes.
To determine if Rb inactivation is sufficient for the induction of hyperplasia or if progression to dysplasia requires some activity in the C-terminus of TAg (independent of p53), I have screened several transgenic lines expressing an amino-terminal mutant of TAg (N136) in villus enterocytes. I found that these mice develop intestinal hyperplasia, although not as early as wild-type TAg does, suggesting that the inactivation of Rb family members is sufficient to induce this phenotype. Furthermore, the appearance of signs of dysplasia was significantly delayed.
I performed global analysis of gene regulation in MEFs and in mouse intestinal epithelium expressing TAg or various mutants. In mouse intestine most of the gene regulation is dependent on binding and inactivation of Rb-proteins by the LXCXE motif and J domain. Regulated genes are involved in cell cycle and proliferation. In MEFs genes belonging to cell cycle, apoptosis and growth factors are differentially regulated by TAg and its mutants. Additionally, we found upregulation of immune response genes by TAg requires the LXCXE motif and some activity mapping to the C-terminus of TAg for their regulation. Significant numbers of genes were found to be regulated independently of the LXCXE motif, J domain and p53 binding domain. This suggests activity independent of these functions.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-02202008-105301 |
| Date | 16 June 2008 |
| Creators | Rathi, Abhilasha Vikas |
| Contributors | PAULA A. GRABOWSKI, JAMES M. PIPAS, BETH STRONACH, BO LIU, JEFFERY L. BRODSKY |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-02202008-105301/ |
| Rights | restricted, 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 Pittsburgh 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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