Apoptosis is characterized by an intracellular proteolytic cascade consisting of a family of proteases called Caspases, and activation of this cascade by extracellular or intracellular stress signals results in the characteristic morphological and biochemical features of apoptotic cell death. The Inhibitors of Apoptosis Proteins (IAPs) are a family of proteins that bind and inhibit Caspases and have been demonstrated to be the only known endogenous inhibitors of the terminal caspase cascade. Defects in apoptosis allow cancer cells to survive under conditions of enormous stress and increased expression of several members of the IAP family has been observed in tumor biopsy samples and cancer cell lines. XIAP Associated Factor 1 (XAF1) was identified as a nuclear protein that binds to the X-linked Inhibitor of Apoptosis protein (XIAP) and antagonizes the anti-caspase activity of XIAP, thereby reversing the protective effect of XIAP overexpression in cancer cell lines. The expression of XAF1 is significantly reduced in cancer cell lines and several primary malignancies as a result of promoter hypermethylation, and re-activation of xaf1 by DNA methylation inhibitors sensitizes cancer cells to apoptosis-inducing agents. Xaf1 has recently been identified as an interferon (IFN)-stimulated gene that sensitizes tumor cells to apoptosis by TRAIL (TNF-related Apoptosis Inducing Ligand) following treatment with IFN-beta.
This thesis documents the role of XAF1 in cell cycle and its potential as a negative regulator of Survivin, an unusual IAP that possesses poor anti-Caspase activity, and is known to be involved in the cell cycle as a member of the chromosomal passenger complex. In HEL 299 and wild-type mouse embryonic fibroblast (MEF) cells, FACS profiling of DNA content suggests that XAF1 overexpression triggers a G1 cell cycle arrest by upregulating p21. This XAF1-mediated upregulation of p21 is not seen in p53-null MEFs and is attenuated by a dominant-negative p53-mutant in HEL 299 cells, suggesting that XAF1 functions primarily upstream of p53 in the cell cycle arrest pathway and results in activation or repression of p53-target genes. Survivin is negatively regulated by p53 and these results suggest that XAF1 may regulate Survivin expression in a p53-dependent manner. Furthermore, overexpression of XAF1 in p53-deficient cancer cells results in significant downregulation of Survivin expression, indicating the existence of a p53-independent mechanism for the observed effect. Immunoprecipitation experiments indicate that XAF1 binds to all the IAPs tested except Survivin. Interestingly, it was observed that XAF1complexes with Survivin only in the presence of XIAP. Furthermore, such an association was associated with a decrease in expression levels of Survivin. XIAP is a RING-bearing protein which functions as an E3 ligase and is involved in the ubiquitin-proteasome pathway. Treatment of transfected cells with proteasomal inhibitor or the use of a XIAP RING-mutant lacking E3 ligase activity was shown to restore Survivin protein expression indicating that XAF1 mediates Survivin downregulation by promoting the E3 ligase activity of XIAP. Taken together, these results suggest that XAF1negatively regulates Survivin expression by both p53-dependent and -independent mechanisms.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/27327 |
| Date | January 2006 |
| Creators | Arora, Vinay |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 93 p. |
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