The impact of folate on telomere length and chromosome stability in human WIL2-NS cells and lymphocytes.

Abstract

Folate is an essential micronutrient required for one-carbon metabolism involved in regulating DNA synthesis, DNA repair and gene expression. Dietary deficiencies in folate result in an increased uracil:thymidine ratio and cytosine hypomethylation in the genome, as well as chromosomal aberrations, the latter being a validated biomarker of cancer risk. Telomeres, the regions of DNA that cap the ends of each chromosome, are critical for maintaining chromosome stability, however, the impact of folate deficiency on telomere structure and function had not previously been investigated. It was hypothesised that the high frequency of thymidine residues in the telomeric repeating hexamer,(TTAGGG)n, may cause this region to be particularly vulnerable to damage caused by folate insufficiency, leading to accelerated telomere attrition if uracil was incorporated into DNA instead of thymidine. In vitro studies were conducted to test this hypothesis using WIL2-NS cells (a p53 deficient B-lymphoblastoid cell line), cultured in medium containing low, medium or high concentrations of folic acid (FA). A flow cytometric method was used to measure telomere length (TL) at regular time points, and these data were correlated against biomarkers of chromosomal instability (CIN) scored in the cytokinesis-block micronucleus cytome (CBMNCyt) assay (micronuclei (MNi), nuclear buds (NBuds) and nucleoplasmic bridges (NPBs)), global hypomethylation and uracil incorporation into telomeric DNA sequences. Findings in the WIL2-NS model showed a significant decline in TL over the longer term (> 14 days of culture), consistent with the hypothesis. In the short term (< 14 days of culture), however, a significant and rapid increase in TL was recorded in low FA cultures, in a dosedependent manner. Furthermore, consistent with previous literature, all biomarkers of CIN increased significantly under low FA conditions. As such, the relationship between TL and CIN was found to be significant and positive in the short term, the opposite to that hypothesised, indicating that the generation of cells with longer telomeres by FA deficiency coincided in a greater degree of CIN during this period. In exploring the mechanism underlying the rapid elongation of telomeres under low FA conditions, new evidence came to light which suggested that hypomethylation of the subtelomere may lead to increased TL. As folate is required for maintenance of DNA methylation, a new hypothesis was then proposed; that hypomethylation due to FA insufficiency results in telomere elongation. This new hypothesis was tested by culturing WIL2-NS cells in complete medium containing a DNA methyltransferase inhibitor, 5-aza-2’ deoxycytidine (5azadC). Results showed a significant, rapid increase in TL with increasing 5azadC, verifying that hypomethylation was the likely cause of telomere elongation observed in this cell type and these events also coincided with large increases in CIN biomarkers. Another novel finding arising from this project was a high frequency of cytokinesis-blocked, binucleated cells displaying multiple NPBs following culture either in low FA, or high 5azadC. New nuclear morphologies, possibly arising from the formation of multiple dicentric chromosomes, were then identified and scored as part of this study. As NPBs can be representative of fusions between chromosomes with compromised telomeres, the high frequencies of these nuclear morphologies suggest that maintenance methylation may play an important role in protecting telomere integrity. Following on from the WIL2-NS studies, peripheral blood lymphocytes (PBLs) were cultured under FA deficient conditions. Results showed that FA concentration had no impact on TL in this cell type, however, significant increases in biomarkers of CIN were observed. Again, novel nuclear morphologies, possibly due to multiple dicentric chromosome formation, were identified in PBL cells cultured under FA deficient conditions. These findings further suggested that folate deficiency may result in enhanced chromosome fusigenic potential. A final investigation was conducted to explore the in vivo relationship between TL of PBL with plasma folate (PF), vitamin B12 (B12) and homocysteine (Hcy) status, and whether any such relationship was dependent on age, gender, body mass index (BMI) and common polymorphisms in folate metabolism genes. Significant relationships were only observed in the older male subset of the cohort whereby plasma folate was found to be positively associated with shorter TL, while TL and plasma Hcy were inversely associated. Overall, the findings of these studies demonstrate that FA deficiency in vitro impacts telomeres differentially, depending on cell type and cell culture duration, and that the hypomethylating effect of low folate may impact telomere integrity indirectly possibly via hypomethylation or other unexplored mechanisms. The findings that short-term folate deficiency and DNA hypomethylation may lead to telomere elongation, in parallel with a dramatic increase in CIN, and specifically multiple NPBs, has not previously been shown. In vivo findings, however, suggest that low folate, and high Hcy, may also have an adverse impact on telomere length, particularly in older males. Most importantly, results of this study show that TL, alone, is probably inadequate and inappropriate as a sole measure of chromosomal instability, and that biomarkers of telomere structure and dysfunction, and possibly subtelomeric DNA methylation, are likely to be of considerably greater value in this context and should be considered for validation in future studies.