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© 2006 American Society for Nutrition J. Nutr. 136:2748-2753, November 2006

Biochemical, Molecular, and Genetic Mechanisms

Global DNA and p53 Region-Specific Hypomethylation in Human Colonic Cells Is Induced by Folate Depletion and Reversed by Folate Supplementation1

Gillian R. Wasson2, Angela P. McGlynn2, Helene McNulty2, Sharleen L. O'Reilly3, Valerie J. McKelvey-Martin2, George McKerr2, J. J. Strain2, John Scott3 and C. Stephen Downes2,*

2 Centre for Molecular Biosciences, University of Ulster, Coleraine, Northern Ireland and 3 Department of Biochemistry, Trinity College, Dublin 2, Ireland

* To whom correspondence should be addressed. E-mail: cs.downes{at}ulster.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
There is increasing evidence to suggest that reduced folate status may be a causative factor in carcinogenesis, particularly colorectal carcinogenesis. Folate is essential for the synthesis of S-adenosylmethionine, the methyl donor required for all methylation reactions in the cell, including the methylation of DNA. Global DNA hypomethylation appears to be an early, and consistent, molecular event in carcinogenesis. We have examined the effects of folate depletion on human-derived cultured colon carcinoma cells using 2 novel modifications to the Comet (single cell gel electrophoresis) assay to detect global DNA hypomethylation and gene region–specific DNA hypomethylation. Colon cells cultured in folate-free medium for 14 d showed a significant increase in global DNA hypomethylation compared with cells grown in medium containing 3µmol/L folic acid. This was also true at a gene level, with folate-deprived cells showing significantly more DNA hypomethylation in the region of the p53 gene. In both cases, the effects of folate depletion were completely reversed by the reintroduction of folic acid to the cells. These results confirm that decreased folate levels are capable of inducing DNA hypomethylation in colon cells and particularly in the region of the p53 gene, suggesting that a more optimal folate status in vivo may normalize any DNA hypomethylation, offering potential protective effects against carcinogenesis. This study also introduces 2 novel functional biomarkers of DNA hypomethylation and demonstrates their suitability to detect folate depletion–induced molecular changes.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The B vitamin, folate, has emerged as an important nutritional factor that may modulate carcinogenesis, particularly colorectal carcinogenesis (CRC). Most published epidemiological and clinical studies have suggested that reduced folate levels are associated with an increased CRC risk (reviewed in 1,2) and evidence from some epidemiological studies (3,4) and a few small intervention trials (5–10) suggests that folate supplementation may reduce CRC risk. Despite recent concerns regarding potential adverse effects of high dose folic acid supplementation on polyp progression (11,12), the majority of studies are consistent with optimal folate status being protective against CRC.

Folate may reduce carcinogenesis through several different mechanisms including the maintenance of normal DNA synthesis and normal methyl group metabolism. Folate is a crucial factor in the synthesis of S-adenosylmethionine, which serves as the methyl donor for methylation of DNA (13). DNA methylation is an important determinant in many cellular processes including gene expression and gene integrity (14). A genome-wide decrease in DNA methylation is one of the earliest molecular abnormalities observed in many human cancers, including CRC (13,15). In human studies using both lymphocyte DNA and colonic mucosa, moderate folate depletion has been shown to correlate with genomic DNA hypomethylation (16–18). Conversely, folate supplementation has been shown to increase DNA methylation in human colonic mucosa (6,8,19). However, other folate intervention trials showed no effect on global DNA methylation (20). On a gene-specific level, folate depletion has been shown to be associated with hypermethylation of the p16 gene in tumor tissue from head and neck squamous cell carcinoma patients (21). Animal studies have shown mixed results; global hypomethylation was found in the livers of rats fed a folate-deficient diet (22), whereas moderate folate depletion showed no significant effect on global and c-myc–specific DNA methylation in rat colonic and hepatic tissue (23,24). Likewise, no significant hypomethylation was found in isolated colonocytes of rats fed a folate-deficient diet (25). In cell culture–based studies, global DNA methylation was found to be reduced in immortalized normal human colonocytes grown in the absence of folate (26) but not in folate-deprived human colon carcinoma cell lines (27), whereas both hypermethylation of the H-Cadherin gene and hypomethylation of the folate-binding protein gene were observed in folate-depleted human nasopharyngeal carcinoma cells (28,29).

Alteration of the p53 tumor-suppressor gene is one of the most frequent abnormalities observed in human cancer and is associated with the transition from adenoma to carcinoma in CRC (30). Both global and p53-specific DNA hypomethylation have been observed in preneoplastic rat hepatic tissue after folate depletion (31,32). This hypomethylation was identified in the same sites within the p53 gene in which an increase in strand breaks has also been found (33). However, in rat hepatic tumor tissue, a relative hypermethylation was found, suggesting that hypomethylation in preneoplasia (and activation of p53) may be followed by de novo methylation and inactivation of the p53 gene as carcinogenesis proceeds (31).

In this study, we have developed specific biomarkers of reduced folate status of relevance to CRC. Using these sensitive and convenient assays, we have examined the effects of folate depletion on both global and p53 gene region–specific DNA hypomethylation in a human colonic cell line.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Cell culture. As there are were no commercially available nontransformed colorectal epithelial cell lines available, the established human colonic adenocarcinoma cell line, SW620, was used. Cells were maintained at 37°C without CO2 in Leibovitz's L15 medium supplemented with 10% dialyzed fetal bovine serum and penicillin/streptomycin. For azacytidine treatment, cells were grown in the presence of 0.5 mg/L 5-azacytidine for 48 h (this concentration of azacytidine has been shown to inhibit DNA methylation by >90% in human colon cells (34). For analysis of folate-depleted colonic cells, cells were grown in folate-free Leibovitz's L15 medium (specially prepared by Invitrogen) or L15 medium containing normal amounts of folic acid (3µmol/L) for 7 or 14 d. For folate repletion experiments, cells were grown in folate-free medium for 7 d and then transferred to L-15 medium containing 3µmol/L folic acid for a further 7 d. Cells were then harvested by trypsinization and processed for the Comet assay.

Cellular folate concentration in these folate-replete and folate-deprived SW620 cells was assessed using the method of Molloy and Scott (35) in which folate was analyzed microbiologically using Lactobacillus rhamnosus in a 96-well microtiter plate after 42 h incubation at 37°C and read at 590 nm spectrophotometrically.

    Methylation-sensitive comet assay. Cells on Comet assay slides were prepared as previously described (36) and immersed in a neutral lysis buffer (2.5 mol/L LiCl, 30 mmol/L EDTA, 10 mmol/L Tris, 0.1% lithium dodecyl sulfate, pH 8.0, 0.03 g/L proteinase K). Slides were incubated at 37°C overnight. The slides were then washed with PBS, and the agarose gel covered with 50 µl of either enzyme buffer alone (NEBuffer IV and 0.1 g/L bovine serum albumin) or 2.5 U/gel HpaII and HhaI restriction enzymes in buffer. The slides were incubated in a moist atmosphere at 37°C for 1 h. Slides were then transferred to alkaline lysis solution (2.5 mmol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Tris, pH 10, and 1% Triton x100) for 1 h at 4°C. Following lysis, unwinding, electrophoresis, and staining with ethidium bromide were carried out as previously described (37).

Comet analysis was carried out using Komet 4.0 software (Kinetic Imaging). The percentage of DNA in the Comet tail for 50 cells was analyzed from each slide and 3 independent experiments were carried out to generate each data point before statistical analysis was carried out. Percentage Comet tail DNA is a measure of the intensity of fluorescence in the Comet tail and reflects the number of strand breaks in the cell. It is defined as 100 – [(head optical intensity/total optical intensity) x 100]. DNA hypomethylation was measured by subtracting the mean percentage of Comet tail from buffer-treated slides (endogenous strand breakage) from the mean percentage Comet tail obtained after incubation with the methylation-sensitive restriction enzymes (strand breakage due to hypomethylated recognition sites). An increase in percentage Comet Tail after enzyme treatment is indicative of DNA hypomethylation as the methylation-sensitive enzymes cause strand breaks at hypomethylated recognition sites, which are then free to move into the Comet tail. Results were expressed as the mean increase in percentage Comet Tail DNA after enzyme treatment. The CV for the assay was 13.4%.

    Methylation-sensitive Comet fluorescent in situ hybridization assay. This assay was carried out using an adaptation of the comet fluorescent in situ hybridization (FISH) procedure from McKelvey-Martin et al. (37) modified to include a methylation-sensitive enzyme digestion step. Comet slides were prepared and processed as described above for the Comet assay for the detection of DNA hypomethylation until the neutralization step. After neutralization, FISH was carried out as previously described (37). The FISH probe used was a 145-kb locus-specific identifier full-length DNA probe for p53 labeled with Spectrum Orange fluorophore (Vysis).

Comet FISH images were obtained using a Nikon Coolpix digital camera. The number and location (i.e. head or tail) of hybridization spots within each Comet was recorded by manual analysis and the percentage spots in the Comet tail calculated. Hybridization of the p53 FISH probe in the Comet tail indicates the presence of a strand break in the region of the p53 gene. Fifty cells were analyzed from each slide and 3 independent experiments were carried out to generate each data point before statistical analysis was carried out. Gene region–specific DNA hypomethylation was measured by subtracting the mean percentage of p53 spots in the Comet tail from buffer-treated slides (endogenous strand breakage in the region of p53 gene) from the mean percentage Comet tail obtained after incubation with the methylation-sensitive restriction enzymes (strand breakage due to hypomethylated recognition sites in the region of the p53 gene). An increase in the percentage of p53 spots appearing in the Comet tail after enzyme treatment is indicative of p53 gene-region hypomethylation. Results were expressed as the mean increase in percentage spots in the Comet tail after enzyme treatment. The CV for the assay was 13.2%.

    Methyl acceptance assay. This assay was carried out as previously described (22,27) and used in this study to validate the results from the methylation-sensitive Comet assay. Each sample was processed in triplicate. Results were expressed as dpm/0.5µg of DNA.

    Statistical methods. All data are presented as mean ± SEM. Data from the initial methylation-sensitive Comet experiment (azacytidine-treated vs. untreated cells) and from the methyl acceptance assay (folate-replete vs. folate-depleted cells, 14 d) were analyzed using the Student's t test. To analyze the data from the methylation-sensitive Comet assays on cells grown in folate-replete or -depleted conditions (folate-replete, 14 d; folate-depleted, 7 and 14 d; and 7 d folate-depleted/7 d folate-replete), 1-way ANOVA was used followed by Tukey's post hoc tests. Equality of variances was confirmed using Levene's test for homogeneity of variances. All statistical analyses were performed with SPSS (version 11.5). P-values < 0.05 were considered significant.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Initial validation of the modified Comet assay using cells treated with the methyltransferase inhibitor, 5-azacytidine. SW620 cells treated with 5-azacytidine had a greater increase (P = 0.017) in mean percentage Comet tail after enzyme treatment compared with untreated cells (5.7 ± 2.6 in untreated cells compared with 15.3 ± 1.6 in azacytidine-treated cells; n = 3 independent experiments). This experiment confirmed that the modified Comet assay using methylation-sensitive enzymes can be used to detect global DNA hypomethylation.

    Assessment of folate levels in cultured colon cells grown in folate-deprived conditions. Cultured colon carcinoma cells were grown for 7 d or 14 d in folate-free medium and compared with cells grown in medium supplemented with normal levels of folic acid. Analysis of the folate levels in these cells by microbiological assay showed that folate depletion for 7 d caused an 81% decrease in cellular folate, whereas growth in folate-free medium for 14 d caused a further significant (P < 0.001) decrease (Fig. 1A). To determine whether the effects of folate depletion could be reversed by supplementation, cells grown in folate-free medium for 7 d were then supplemented with 3µmol/L folic acid for a further 7 d. After this repletion period, cellular folate levels had reached 83% of normal cellular folate but had not completely returned to their predepletion levels.


Figure 1
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  		Figure 1  Cellular folate levels (A), and global (B) and gene-specific DNA hypomethylation (C) in SW620 human colon carcinoma cells grown in folate-replete medium (+F14), or under folate-depleted conditions for 7 d (–F7) or 14 d (–F14), or under folate-depleted conditions for 7 d before supplementing with folic acid for a further 7 d (–F/+F). Results are means ± SEM (n = 3 independent experiments). Means without a common letter differ, P < 0.05.

 
    Detection of global DNA hypomethylation in folate-deprived colon cells. An increase in global DNA hypomethylation was seen in folate-depleted colon cells, as evidenced by an increase in percentage Comet tail after digestion with the methylation-sensitive endonucleases compared with cells grown in folate-replete medium (Fig. 1B). This increase in hypomethylation was not significant in cells grown in folate-free conditions for 7 d (P = 0.288), but by 14 d, hypomethylation was found to be increased compared with folate-replete cells (P = 0.014; Fig. 1B). In the cells that underwent folate depletion and then repletion, global DNA hypomethylation levels were the same as cells continually grown in folate-rich medium (Fig. 1B).

    Analysis of p53 gene-specific DNA hypomethylation in folate-deprived colon cells. Results from the methylation-sensitive Comet FISH assay indicated that specific DNA hypomethylation within the region of the p53 gene was occurring during folate depletion. Cells grown in folate-free conditions for either 7 or 14 d showed a greater increase (P = 0.010 for 7 d and P = 0.027 for 14 d) in the percentage of FISH spots in the Comet tail in slides treated with methylation-sensitive endonucleases compared with folate-replete cells (Fig. 1C and Fig. 2). An increase of the percentage of spots in the Comet tail is indicative of hypomethylation in the region of the p53 gene resulting in digestion of the DNA with the methylation-sensitive enzymes and movement of the DNA loop containing the p53 gene into the Comet tail. As before, cells that were folate deprived and then supplemented showed similar levels of hypomethylation to folate-replete cells (Figs. 1C and 2).


Figure 2
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  		Figure 2  Images of methylation Comet-FISH taken from SW620 colon cells grown in: folate-depleted medium for 7 d (A,B); folate-depleted medium for 14 d (C,D); folate-replete medium (E,F); and folate-depleted medium for 7 d followed by folate-replete medium for 7 d (G,H). Comets were either treated with enzyme buffer alone (A,C,E,G) or with methylation sensitive endonucleases (B,D,F,H) to detect specific DNA hypomethylation of the p53 gene region. An increase in the percentage of p53 gene probe spots appearing in the Comet tail indicates increased DNA hypomethylation of the p53 gene region. DNA is labeled with 4'6-diamino-2-phenylindole (blue) and the p53 probe is labeled with Spectrum Orange (red spots).

 
    Global DNA methylation levels in folate depleted colon cells measured using the methyl acceptance assay. This assay exploits the fact that the ability of DNA to incorporate exogenous [3H-methyl] groups is inversely related to endogenous DNA methylation. Using this assay, an increase in global DNA hypomethylation was observed in cells grown for 14 d in folate-free conditions compared with cells grown in folate-replete conditions, as shown by greater [3H-methyl] incorporation (P = 0.020) into folate-depleted cells (38700 ± 991 dpm/0.5µg DNA for folate-depleted cells compared with 33100 ± 2310 dpm/0.5µg DNA for folate-replete cells, n = 6, representing 2 independent experiments carried out in triplicate).


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Studies indicate that up to 30% of healthy populations may have subclinical but biochemically evident degrees of reduced folate status (38). There is increasing evidence that even small reductions in folate levels, not sufficient to cause an overt clinical manifestation (i.e. megaloblastic anemia), may be implicated in a range of disease states, including neural tube defects, cardiovascular disease, and cancer (39,40). The mechanisms through which such reduced folate status may influence these conditions have not been fully elucidated. In this study, techniques were developed and optimized to assess 2 specific molecular biomarkers relevant to folate, global DNA hypomethylation, and p53 gene region–specific hypomethylation.

The results obtained from this study support the hypothesis that impaired folate status leads to global and gene-specific DNA hypomethylation. A modified Comet assay was developed that incorporated the methylation-sensitive restriction enzymes, HpaII and HhaI, which cut the DNA only at unmethylated recognition sites. The validity of the assay was initially confirmed using cultured cells treated with 5-azacytidine, which induces hypomethylation by inhibition of the DNA methyltransferase. Folate-depleted colon carcinoma cells were then assessed for global DNA hypomethylation and found to have increased levels of hypomethylation compared with folate-replete cells. This increase in DNA hypomethylation had not reached significance by 7 d, despite a decrease in cellular folate levels of >80% at this time. However, by 14 d, a significant increase in DNA hypomethylation was detected and cellular folate level was reduced to one-half of the level at 7 d. We also observed that cells grown under folate-depleted conditions for 7 d and then returned to folate-replete medium (to mimic the effects of folate supplementation) showed similar levels of hypomethylation to cells grown in folate-replete conditions throughout the experimental period (Fig. 1B). These results suggest that global DNA hypomethylation may be a dose-dependent response to folate deprivation that can be readily corrected by folate supplementation. DNA methylation is known to play many important roles in the cell, including roles in regulation of gene expression, conformational configuration and structural stability of DNA, binding of transcription factors and other proteins, mutagenesis, and imprinting (41). Our finding is in general agreement with previous studies showing that genomic and gene-specific DNA hypomethylation appears to be a consistent event in carcinogenesis (42,43), including that of colorectal cancer (44,45).

The global DNA methylation results obtained using the methylation-sensitive Comet assay were confirmed in this study using the methyl acceptance assay for DNA methylation (22,27). This assay is based on the principle that an inverse relation exists between endogenous DNA methylation status and exogenous [3H-methyl] incorporation. Using this approach, we demonstrated that cells grown for 14 d in folate-free conditions had a greater incorporation of [3H-methyl] compared with folate-replete cells (P < 0.05), indicating reduced DNA methylation in the folate-depleted cells and confirming the above results using the methylation-sensitive Comet Assay. In contrast to the current finding, Stempak et al. (27) reported no significant increase in DNA hypomethylation in response to folate depletion in the colonic adenocarcinoma cell lines, HCT116 and Caco-2, using the methyl acceptance assay. However, closer examination of their data reveals that a 7% increase in exogenous [3H-methyl] incorporation was found in both cell lines. Although these increases were not significant (P = 0.162 for HCT116 and P = 0.128 for Caco2), they are comparable to the 14% increase observed in SW620 cells in our study. Duthie et al. (26) also used the methyl acceptance assay to measure global DNA hypomethylation and found a significant increase in hypomethylation in immortalized normal human colonocytes grown for 14 d in the absence of folate.

The folate deficiency–induced global DNA hypomethylation found in SW620 cells in this study was mirrored at a gene-specific level. Incorporation of a FISH step using a fluorescently labeled probe specific for the p53 gene region allowed the methylation-sensitive Comet assay to be used for the detection of p53 region-specific DNA hypomethylation. Using this assay, we found an increased percentage of p53 gene region hybridization spots appearing in the Comet tails of folate-depleted cells after enzyme treatment compared with cells grown in folate-replete conditions. These results indicate that there were more hypomethylated sites in the region of the p53 gene in the folate-deprived cells. However, due to the large size of the commercial probe used (145 kb) and the nature of the Comet assay, it is not possible to definitively conclude that the hypomethylated sites occur within the p53 gene itself. What the results do show is that the hypomethylated sites occur in the loop of DNA containing the p53 gene, findings that are consistent with previous studies showing p53-specific DNA hypomethylation in folate-depleted rat hepatic tissue (31,32). In our study, maximal gene region–specific hypomethylation was reached by 7 d and further folate depletion did not cause a further increase, unlike global DNA hypomethylation. These results suggest that the HpaII/HhaI restriction sites in the p53 gene region may be fully unmethylated by 7 d, because further folate deprivation does not have any additional effect. Therefore, p53 gene region–specific methylation appears to be more sensitive to folate status than global DNA methylation. Folate depletion followed by repletion resulted in similar p53 region-specific hypomethylation levels to cells grown continually in folate-replete conditions (Figs. 1C and 2). As de novo methylation of CpG islands in gene promoter regions is associated with gene silencing (46), gene-specific DNA hypomethylation may contribute to carcinogenesis through the activation of growth promoter genes and oncogenes. DNA methylation is also important in the conformational configuration and structural stability of DNA (47). Regional hypomethylation of genes may promote strand breaks and mutagenesis through alterations in chromatin conformation, which increase the accessibility of the DNA to DNA-damaging agents, thereby promoting genomic instability (48,49).

Apart from the findings discussed above, this study also presents the development and validation of simple and sensitive techniques capable of analyzing both global and gene region–specific DNA hypomethylation at a single cell level for wider application in nutrition and cancer research. Traditional methods for detecting global DNA methylation such as HPLC-mass spectrometry, bisulfite sequencing, or methylation-sensitive microarrays, necessitate either the use of expensive and complex equipment or a high degree of technical expertise and generally require large numbers of cells as starting material (50). Other methods such as the methyl acceptance assay and the cytosine extension assay (51) require the use of radioactive material. In contrast, the methylation-sensitive Comet assay is a simple and relatively inexpensive way to analyze DNA methylation in a small number of cells, and on a cell-by-cell basis rather than on a heterogeneous population. In particular, the methylation-sensitive Comet FISH assay allows the analysis of a gene region without complicated primer design or the need to rely on PCR amplification or bisulfite modification. These assays are suitable for use as functional biomarkers to assess the molecular effects of folate deficiency relevant to carcinogenesis. We are currently employing these techniques as endpoints in a folate intervention trial in patients with colorectal polyps.

In conclusion, results from this study demonstrate, to our knowledge for the first time, p53 gene region–specific DNA hypomethylation in response to folate depletion in human cells and confirm previous work suggesting that folate depletion leads to global DNA hypomethylation. This study also shows that folate supplementation of folate-deprived cells may return DNA methylation levels to normal. As the molecular effects of folate levels, and in particular the effects of folate supplementation, remain the subject of much debate (11,12), the implications of our findings will be important to further elucidate these effects and add evidence to the debate on the advantages of food policy strategies such as folic acid fortification.


   FOOTNOTES
 
1 Supported by contract N05028 from the Food Standards Agency (FSA), UK. Back

Manuscript received 18 May 2006. Initial review completed 22 June 2006. Revision accepted 1 September 2006.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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G. R. Wasson, V. J. McKelvey-Martin, and C. S. Downes
The use of the comet assay in the study of human nutrition and cancer
Mutagenesis, May 1, 2008; 23(3): 153 - 162.
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