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RFA CA-03-016:
Diet, DNA Methylation and Other Epigenetic Events, and Cancer Prevention

Dietary Affects Upon Genomic Methylation in Trout Cancer

Principal Investigator: Brunelli, Joseph P
Institution: Washington State University
NCI/DCP Program Director: Ross, Sharon A
Project ID (Grant #): 1R21CA105293-01
RFA/PA Number: RFA-CA-03-016
Project Funding Period: 05/28/2004 to 04/30/2006

Description (provided by applicant)

The goal of this project is to characterize the effect of dietary supplements expected to impact epigenetic methylation of genomic regions, and associate the variability in methylation to development of nephroblastomas and hepatocarcinomas through exposure to mutagens in clonal lines of rainbow trout. Trout are uniquely suited for this study because of their large size, their sensitivity to mutagens, the well developed research program exploring dietary impacts upon cancer formation, the availability of clonal lines and the ongoing development of a good comparative map in this species. Extensive evidence from studies in mammals demonstrates that the development of cancer involves epigenetic mechanisms affecting gene expression through aberrant genomic methylation. These changes to the genomic methylation status can also develop in response to dietary regimens affecting availability of biochemical substrates to metabolic pathways controlling CpG methylation. Furthermore, this process appears to include a heritable influence through a dietary impact upon the imprinting of gametes. Trout offer an opportunity to analyze, at low cost, candidate dietary elements capable of impacting cancer formation by experimentally controlling dietary variables and tumor development, in a genetically defined background of clonally derived isogenic populations. Specifically, this investigation is designed to reveal correlations between cancer incidence and dietary influences upon aberrant epigenetic CpG methylation causing gene silencing. Project objectives include: 1) Conducting genome-wide screens of CpG methylation patterns characterizing differentiated tissues recovered from isogenic juvenile trout, using the Methylation Sensitive-Amplified Fragment Length Polymorphism (MS-AFLP) technique to determine if genomic methylation patterns are influenced by dietary regimens which manipulate the availability of (folate, methionine, choline, vitamins B12, betaine and zinc). 2) Characterize nephroblastoma and hepatocarcinoma incidence and methylation polymorphisms in isogenic populations exposed to carcinogens and grown under dietary regimens demonstrated to alter methylation. 3) Analyze genome sequences of common MS-AFLP polymorphisms found in cancers, verify tumor associated methylation polymorphisms reflect physiologically relevant epigenetic events, by methylation-specific PCR and bisulfite sequencing to reveal normal tissue methylation patterns, compared to affected loci. Compare expression of genes found to be linked to tumor associated MS-AFLP sites, between normal and tumor tissues, using RT-PCR. 4) Integrate mapable diet associated MS-AFLP polymorphisms into the existing OSU x Arlee map. This investigation will provide fundamental insights into toxicological epigenetic influences through altered genomic methylation and gene expression, and allow the development of a genome-wide tissue-specific methylome profile using an experimentally tractable lower vertebrate model system. Pullman, WA 99164-4236.

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