Folate Deficiency In Vitro Induces Uracil Misincorporation and DNA Hypomethylation and Inhibits DNA Excision Repair in Immortalized Normal Human Colon Epithelial Cells

Abstract

Epidemiological studies have indicated that folic acid protects against a variety of cancers, particularly cancer of the colorectum. Folate is essential for efficient DNA synthesis and repair. Moreover, folate can affect cellular S-adenosylmethionine levels, which regulate DNA methylation and control gene expression. We have investigated the mechanisms through which folate affects DNA stability in immortalized normal human colonocytes (HCEC). DNA strand breakage, uracil misincorporation, and DNA repair, in response to oxidative and alkylation damage, were determined in folate-sufficient and folate-deficient colonocytes by single cell gel electrophoresis. In addition, methyl incorporation into genomic DNA was measured using the bacterial enzyme Sss1 methylase. Cultured human colonocyte DNA contained endogenous strand breaks and uracil. Folate deficiency significantly increased strand breakage and uracil misincorporation in these cells. This negative effect on DNA stability was concentration dependent at levels usually found in human plasma (1-10 ng/ml). DNA methylation was decreased in HCEC grown in the absence of folate. Conversely, hypomethylation was not concentration dependent. Folate deficiency impaired the ability of HCEC to repair oxidative and alkylation damage. These results demonstrate that folic acid modulates DNA repair, DNA strand breakage, and uracil misincorporation in immortalized human colonocytes and that folate deficiency substantially decreases DNA stability in these cells.

Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and colorectal cancer: The Fukuoka Colorectal Cancer Study

“…An adequate pool of thymidylate decreases deoxyuridylate-induced DNA damage and ensures efficient DNA synthesis and repair. [3][4][5] In this regard, inconsistency in the association with MTHFR 677TT genotype among studies may be related to different folate levels in different populations. Folate intake seems fairly high among adults in Japan; the average intake was estimated to be 330 µg per day in the National Nutrition Survey in 2001.…”

“…3,4) Furthermore, insufficient thymidylate can increase DNA misrepair, resulting in overall DNA damage in the cell. 5) On the other hand, 5-methyltetrahydrofolate provides the methyl group for methylation of homocysteine to methionine. Imbalanced DNA methylation, i.e., global genomic hypomethylation and methylation of usually unmethylated CpG sites, has been implicated in colorectal carcinogenesis.…”

Diet and cancer prevention

“…Low folate (in the form of 5-methyltetrahydrofolate) status, whether by inadequate intake or metabolism, reduces intracellular S-adenosylmethionine (SAM), and alters cytosine methylation in DNA, potentially leading to inappropriate activation of proto-oncogenes (Duthie, 1999) or inactivation of tumor-suppressor genes. Further, when levels of 5,10-methylenetetrahydrofolate are low, misincorporation of uracil for thymidine occurs during DNA synthesis (Wickramasinghe, 1994;Blount, 1997), increasing the need for DNA repair, which is also compromised when folate is limited (Duthie et al, 2000). These abnormalities can be reversed with folic acid supplementation.…”

Moderate folate depletion modulates the expression of selected genes involved in cell cycle, intracellular signaling and folate uptake in human colonic epithelial cell lines

“…In addition to disturbing nucleotide metabolism, folate depletion is reported to cause genomic hypomethylation in humans [19] and in cell culture [29] and p53 gene-specific hypomethylation in rats [23,24]. Although a depletion of the universal methyl donor Sadenosylmethionine is observed during folate depletion, the accumulation of its precursor Sadenosylhomocysteine, an inhibitor of DNA methyltransferases, appears to be a stronger predictor of hypomethylation [30].…”