The major activity in our laboratory is studies of DNA methylation in tumorigenesis, a process commonly observed in GC-rich sequences called CpG islands in many types of human cancers and is often associated with transcriptional silencing. Using non-Hodgkin's lymphoma (NHL) as a model system, our discovery-driven preliminary studies demonstrate that DNA hypermethylation is not a random event; many CpG island loci are susceptible to methylation alteration. As a result of this epigenetic mutation, the expression of genes that govern key functions of the cell may become silent, leading to clonal proliferation of tumor cells. Differential susceptibility of critical CpG island loci to DNA hypermethylation may therefore influence the development of different NHL subtypes and may help explain differences in tumor growth and treatment outcomes. We have identified several loci that are differentially methylated and may be involved in lymphomagenesis. Our Central Hypothesis: B-cell differentiation is affected by methylation of CpG islands and this frequently leads to silencing of gene transcription. We further hypothesize that 1) Histological classes of NHL actually contain more than one clinical disease; 2) Hypermethylation of CpG island loci in NHL cells can generate unique molecular signatures that are associated with clinical subtypes and; 3) Dissecting these complex epigenetic profiles requires an understanding of gene methylation in normal, as well as neoplastic, B-cell differentiation. This application will expand a current version of our Methylation-Specific Oligonucleotide (MSO) microarray, an invention that combines the power of the bisulfite treatment protocol with the versatility of oligonucleotide microarrays, and apply this innovative technique to study DNA methylation in cases of B-cell NHL and normal B-cells at similar stages of differentiation, and relate these changes to gene silencing and classification. We plan to test our hypotheses by pursuing 4 specific aims; 1. Generate an MSO microarray for analysis of promoter hypermethylation at about 4,000 loci in 80 genes; 2. Determine patterns of CpG island methylation that characterize subsets of NHL classes and their putative normal stage of B-cell differentiation; 3. Correlate the status of promoter hypermethylation defined by MSO with gene expression; 4. Develop and apply data management, analysis and visualization tools to decipher methylation profiles of NHL classes. The proposed studies are expected to yield important insights into potential mechanisms of DNA methylation-driven gene silencing related to B-cell differentiation and development of clinical subtypes of NHL.
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