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      • HARVEST
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      Aberrant epigenetics in the molecular pathogenesis of human acute myeloid leukemia

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      Date
      2005-05-18
      Author
      Scott, Stuart Alexander
      Type
      Thesis
      Degree Level
      Doctoral
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      Abstract
      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.
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Pathology
      Program
      Pathology
      Supervisor
      DeCoteau, John
      Committee
      Xiang, Jim; Saxena, Anurag; Qureshi, Mabood; Massey, K. Lorne; Dong, Wei-Feng; Banerjee, Diponkar
      Copyright Date
      May 2005
      URI
      http://hdl.handle.net/10388/etd-05242005-153230
      Subject
      histone modification
      p15INK4B
      5-Aza-2'-deoxycytidine
      zebularine
      cDNA microarray
      metallothionein
      promoter hypermethylation
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