Investigation of the effects of folic acid deficiency on colon carcinogenesis

GR Wasson, BSca, AP McGlynn, PhDa, SR O'Reilly, BScb, VJ McKelvey-Martin, PhDa, JJ Strain, PhDa, DG Weir, MDb, JM Scott, PhD, ScDb, CS Downes, PhDa and H McNulty, PhDa

aSchool of Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry, BT52 1SA, N. Ireland. bDepartments of Biochemistry and Clinical Medicine, Trinity College, Dublin, Ireland

Aim Folic acid deficiency has been implicated as a risk factor for colon cancer. Folate is required for DNA synthesis and repair and also for DNA methylation reactions, which influence gene expression. We have investigated two biomolecular markers of folic acid deficiency - DNA methylation and the misincorporation of uracil into DNA. Methods Modified Comet assays were used to analyse methylation status and uracil misincorporation in a colon cell line grown in folate-replete and -deplete conditions and also in epithelial cells derived from colonic biopsies collected from patients at risk of colorectal cancer. Combined fluorescent in situ hybridisation (FISH) and methylation-Comets allowed the detection of p53 gene-specific hypomethylation. Results An increase in global DNA and p53 gene-specific hypomethylation and an increase in uracil misincorporation were found in folate depleted cultured cells and also in human biopsies. Conclusions The modified Comet assays described have been optimised for the detection of folate-sensitive biomolecular markers in both a colon cell line model and also in cells derived from small colonic biopsies from patients at risk of colorectal cancer. These assays are now being used to determine biomolecular effects of folate in an intervention patient study.

KEY WORDS: Comet Assay, DNA methylation, Uracil Misincorporation.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Risk Assessment, Part 1.