Promoter hypermethylation mediated gene silencing is a frequent epigenetic finding in many cancers that affects genes known to have important roles in several aspects of cell biology. Hematological malignancies have been reported to harbor multiple genes aberrantly silenced by promoter hypermethylation and as a result, cytosine analogs known to inhibit the DNA methylation machinery are currently being evaluated in clinical trials. As such, the general goal of this thesis was to identify genes silenced by promoter hypermethylation in human acute myeloid leukemia (AML) and to study the mechanism of promoter hypermethylation mediated gene silencing. Interestingly, the cyclin dependent kinase inhibitor p15 was found to be methylated at a high frequency in AML patients and cell lines in association with a lack of detectable p15 mRNA. Treatment with the cytosine analog 5-Aza-2-deoxycytidine (5-Aza-dC) in vitro resulted in promoter demethylation and p15 mRNA re-expression, which was associated with a release of a transcriptionally repressive complex at the p15 promoter. Importantly, 5-Aza-dC treatment also reversed specific histone amino-terminal modifications at the p15 promoter which are normally associated with transcriptionally inactive chromatin regions, implicating chromatin remodeling in promoter hypermethylation mediated gene silencing. The recently discovered DNA methylation inhibitor, zebularine considered more stable than 5-Aza-dC was also able to reconstitute p15 mRNA in vitro in association with promoter demethylation, regional enrichment of histone acetylation, and growth inhibition.
To identify novel genes silenced by promoter hypermethylation in AML, cDNA microarray analysis was employed following in vitro pharmacological inhibition of DNA methylation and histone deacetylation. Of note, four genes from the metallothionein family of cysteine rich small molecules were consistently upregulated following drug treatment and further evaluation identified the gene MT1H to be hypermethylated at a high frequency in AML patients and cell lines. Taken together, the data suggests that aberrant promoter hypermethylation mediated gene silencing occurs in multiple genes from different gene families during the molecular pathogenesis of human AML. Furthermore, the mechanism of promoter methylation mediated transcriptional silencing acts in concert with specific histone modifications which, importantly, can be reversed by treatment with pharmacological inhibitors of DNA methylation.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-05242005-153230 |
| Date | 30 May 2005 |
| Creators | Scott, Stuart Alexander |
| Contributors | Xiang, Jim, Saxena, Anurag, Qureshi, Mabood, Massey, K. Lorne, Dong, Wei-Feng, DeCoteau, John, Banerjee, Diponkar |
| 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-05242005-153230/ |
| 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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