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The establishment of DNA replication biomarkers in colon cells and their validation as functional markers of folate status

Project Code: N05028

30/11/2004

University of Ulster
McNulty, H ; McGlynn, A; Wasson, G; O'Reilly, S; Bradbury, I; Molloy, A; Ward, M; Mckelvey-Martin, V; Mahmud, N; McKerr, G; Strain, J; Scott, J; Downes, C

There is increasing evidence to suggest that folate deficiency may be a causative factor in carcinogenesis, particularly colorectal carcinogenesis. Modification of DNA including misincorporation of uracil, leading to genetic instability, and a reduction in global DNA methylation, promoting aberrant gene expression and chromatin instability, have been proposed as mechanisms by which folate deficiency is implicated in colon carcinogenesis. The overall aim of this project was to adapt existing Comet assay technology to measure these DNA modifications in colonocytes isolated from biopsies of colonic mucosa and to establish such modifications as putative biomarkers of folate status in patients at risk of developing colorectal cancer.

Objective 1 of the study sought to optimise procedures for isolating pure epithelial cells from small colonic biopsies from human colon. Methods for measurement of colonocyte folate together with measurement of the putative DNA biomarkers in these colonocytes by modified Comet assay were also established.

The ion-chelation method for colonocyte isolation from small human biopsies removes the need for enzymatic disaggregation of biopsy tissue and hence produces a high yield of cells with minimal DNA damage, suitable for use with the Comet assay. Close collaboration between our centres in Dublin and Coleraine led to the establishment of a protocol that allowed for optional storage of biopsies in Dublin andsafe transfer of these colonocytes to Coleraine. For the latter, colonocytes were embedded in agarose on Comet glass slides and immersed in neutral lysis buffer for cell lysis and de-proteination prior to transport to Coleraine in the same lysis buffer.
These conditions of transfer meant that no added DNA damage was caused during transfer, particularly in the event of courier delay. At this stage, a study was undertaken to determine which assay of colonic folate, whether in whole biopsies or in isolated colonocytes, is best predicted by systemic folate. Tissue biopsy samples were isolated from the descending colon of 31 individuals following a normal colonoscopy. Blood samples were obtained for the determination of total plasma homocysteine by immunoassay and red cell folate, serum folate and serum vitamin B12 by microbiological assay. Colonocyte folate was also analysed by microbiological assay following the deconjugation of the folate polyglutamate to the monoglutamate form. Although both the whole tissue and colonocyte methods were shown to be useful for examining the relationship between systemic and localised tissue folate status, the isolated colonocyte folate was more accurately predicted by systemic folate status (manuscript 1). Colonocyte folate measurements were therefore used for the rest of the project.

Objective 2 of the study aimed to establish modified Comet assays for uracil misincorporation into DNA and hypomethylation in global DNA and in specific gene regions. These novel assays were modified to be suitable for use with colonocytes isolated from small colonic biopsies. The Comet assay was originally designed to measure DNA damage in single cells by embedding them in agarose and lysing the cells prior to DNA unwinding and electrophoresis. Modifications of the Comet assay have previously been described and were applied here for the detection of misincorporated uracil into DNA. As part of this project, we have developed a novel methylation-sensitive Comet assay to detect global DNA hypomethylation. In addition, we further developed this technique using fluorescence-in-situ-hybridisation (Comet-FISH), to allow the detection of hypomethylation in the region of a specific gene. Colon cells cultured in folate-free medium showed a significant increase in global and p53 gene-specific DNA hypomethylation compared to cells grown in medium containing folic acid. The results of these pilot experiments confirmed that our methylation-sensitive Comet assays were capable of detecting DNA hypomethylation in colon cells and particularly in the region of the p53 gene (manuscript 2, Appendix II). Initial experiments confirmed that both uracil misincorporation and global DNA hypomethylation were measurable in colonocytes isolated from human colonic biopsies at colonoscopy and that both assays were reproducible at multiple regions of normal colon.

Objectives 3 and 4 aimed to investigate the variation of both DNA biomarkers, uracil misincorporation and global DNA hypomethylation, in colon cells isolated from patients with and without polyps, and to investigate an association of these biomarkers with systemic and colonocyte folate status in these patients. These investigations were achieved by means of a case-control study in which a cohort of cases (n=56) diagnosed with either hyperplastic (n=16) or adenomatous (n=40) colonic polyps were recruited, together with a cohort of controls without colonic polyps (n=64). Biopsies were taken from three anatomical sites of the colon (ascending, transverse and descending) in controls, and from the polyp, an area adjacent to the polyp and from an area 10-15cm distal to the polyp in cases. Polyps were found in ascending, transverse or descending colon. Case-control comparisons were evaluated according to anatomical site using a statistical mixed model. Data were adjusted for age and smoking as both were found to be associated with polyp occurrence. Both hyperplastic polyp and adenoma cases displayed lower colonocyte folate than controls (p<0.001). Uracil misincorporation was higher in the adenomas compared with either hyperplastic polyp cases (p<0.001) or controls (p<0.001), whereas no differences were found in global DNA hypomethylation between cases and controls (Manuscript 3). An intervention study was designed to investigate the effect of low dose folic acid supplementation on systemic and colonocyte folate status, and on the putative DNA biomarkers: uracil misincorporation, global DNA and gene-specific hypomethylation (Objective 5). Twenty individuals harbouring colonic adenomas were randomised to receive folic acid (600μg daily) or placebo for 6 months post polypectomy, at which time they were rebiopsied. Folate status (systemic and colonocyte) and DNA biomarkers were determined at baseline colonoscopy and following the 6-month intervention period. Colonocyte folate in the polyp site showed a strong trend towards increased values in the supplemented group (p=0.071) while the normal colon distal to the polyp site showed no response to
supplementation. Similarly, the polyp site post intervention showed a strong trend towards decreased levels of uracil misincorporation (p=0.081), while again the distal site showed no significant change. Neither global DNA hypomethylation nor genespecific hypomethylation showed any significant response to supplementation (Manuscript 4).

Key Findings: Compared with DNA hypomethylation, uracil misincorporation was more indicative of variations in colonic cell folate and polyp type. In the small intervention group studied, folic acid supplementation showed strong trends towards increased folate content of colonocytes and correspondingly decreased levels of uracil misincorporation in colonic mucosa that was adjacent to a colonic adenoma. There was no response in DNA hypomethylation to supplementation.

Conclusions: The results (of both the case-control and the intervention studies) point towards uracil misincorporation in DNA, rather than DNA hypomethylation, as a more informative and sensitive biomarker of low folate status in colorectal carcinogenesis. This can now be confirmed in a larger cohort of patients using a range of folic acid doses and using the methodologies developed here.

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