1 Department of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada,
2 Department of Nutritional Sciences, University of Toronto, Toronto, ON M5S 1A8, Canada,
3 Division of Gastroenterology, St Michaels Hospital, University of Toronto, Toronto, ON M5B 1W8, Canada,
4 Nutrition and Cancer Prevention Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111, USA and
5 Divisions of Clinical Nutrition and Gastroenterology, Department of Internal Medicine, New England Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA
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Abstract |
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Abbreviations: CpG, cytosine-guanine dinucleotide sequences; DMH, dimethylhydrazine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine
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Introduction |
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To date, the mechanisms by which folate deficiency enhances, and supplementation suppresses, colorectal carcinogenesis have not been clearly elucidated (1,2). One proposed mechanism suggests that folate deficiency may induce DNA hypomethylation (1,2). Folate, in the form of 5-methyltetrahydrofolate, is involved in remethylation of homocysteine to methionine, which is a precursor of S-adenosylmethionine (SAM), the primary methyl group donor for most biological methylations, including that of DNA (Figure 1) (10). After transfer of the methyl group, SAM is converted to S-adenosylhomocysteine (SAH), a potent inhibitor of most SAM-dependent methyltransferases (Figure 1
) (10).
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Diets deficient in different combinations of methyl group donors (choline, folate, methionine and vitamin B12) have been consistently observed to induce genomic and protooncogene DNA hypomethylation and elevated levels of corresponding mRNA (1722) and site-specific p53 hypomethylation (2224) in rat liver. However, conflicting data exist for the effect of isolated folate deficiency on DNA methylation in rodent liver (6,2527). Furthermore, the effect of isolated folate deficiency on DNA methylation in the colorectum, the primary target tissue that is particularly susceptible to the folate deficiency-associated carcinogenic effect, has not yet been clearly established.
The present study therefore investigated the time-dependent effect of an isolated dietary folate deficiency and supplementation on DNA methylation at the genomic level and within the promoter region and exons 67 of the p53 tumor suppressor gene in rat colon in order to clarify this issue. We also investigated how changes in promoter and coding region p53 methylation are related to steady-state levels of p53 transcript. Exons 67 of the p53 gene were chosen because previous animal studies have shown that this hypermutable coding region is particularly susceptible to the hypomethylating effect of dietary deficiency of folate alone (27) or combined methyl donors (2224) in rat liver. In the methyl deficiency rat model of hepatocarcinogenesis, the degree of p53 methylation within this coding region was shown to be reciprocally related to the steady-state level of p53 mRNA (24). Furthermore, in the same model, specific CpG sites within exons 67 of the hepatic p53 gene were resistant to demethylation while other CpG sites underwent progressive demethylation in response to methyl deficiency (23). Although the promoter region of the rat p53 gene does not constitute a CpG island, there are 15 CpG sites within the 418 bp region between nt 514 and 92 including the 85 bp minimal essential promoter region (between nt -216 and -131) (28,29). In the methyl deficiency rat model of hepatocarcinogenesis, a single CpG site at nt -450 was determined to be a critical site for initiation of de novo methylation and progressive spreading of methylation associated with transcriptional inactivation of the p53 gene (29).
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Materials and methods |
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Rats were housed individually in wire-bottomed stainless steel cages to minimize coprophagy. Body weights were recorded weekly. Water was supplied ad libitum. The amount of diet supplied to each group was matched to the mean daily food consumption of the group with the least food consumption on a weekly basis. Five pre-assigned rats from each group were killed by exsanguination under carbon dioxide anesthesia weekly for 5 weeks after the dietary regimen began.
Sample collection
Blood was collected into evacuated tubes containing EDTA and centrifuged at 800 g for 10 min at 4°C, and plasma was stored at -70°C in 0.5% ascorbic acid for plasma folate assays. Aliquots (100 µl) of plasma were stored without ascorbate for homocysteine assays. Blood samples for complete blood counts were collected into tubes containing sodium EDTA and analyzed immediately (System 9000; Serono Baker Diagnostic, Allentown, PA). The colorectum was excised and put on a glass plate suspended on crushed ice. The colorectum was opened longitudinally and rinsed in 0.9% NaCl. The mucosa was carefully scraped with glass slides. The resulting colonic mucosal scrapings were rapidly weighted, frozen in liquid nitrogen and stored at -70°C for subsequent extractions of colonic DNA, RNA, folate and protein. Fresh colonic mucosal scrapings were immediately homogenized in 2 vol of 0.4 M perchloric acid at the time of killing and centrifuged at 1500 g for 10 min, and then the resulting supernatant was frozen at -70°C for subsequent analyses of colonic SAM and SAH concentrations.
Folate, homocysteine, SAM and SAH assays
Plasma folate concentrations were determined by a standard microbiologic microtiter plate assay using Lactobacillus casei (36). Colonic mucosal folate concentrations were measured by the same microbiologic assay (36) after tissue folate extraction and subsequent treatment with chicken pancreas conjugase as described previously (37). Colonic mucosal protein concentrations were determined by the methods of Lowry et al. (38). Total plasma homocysteine was measured by HPLC according to the fluorometric method of Vester and Rasmussen (39). Colonic mucosal SAM and SAH concentrations were determined by HPLC with ultraviolet detection (40).
DNA extraction
The DNA from the colonic mucosa was extracted by a standard DNA extraction kit (Easy-DNA; Invitrogen, San Diego, CA) according to the manufacturers protocol. The size of DNA estimated by agarose-gel electrophoresis was >20 kb in all instances. No RNA contamination was detected on agarose-gel electrophoresis. The final preparations had a ratio of A260:A280 between 1.8 and 2.0. The concentration of each DNA sample was determined as the mean of three independent spectrophotometric readings.
Genomic DNA methylation
The methylation status of CpG sites in genomic DNA from the colon was determined by the in vitro methyl acceptance capacity of DNA using [3H-methyl]SAM as a methyl donor and a prokaryotic CpG DNA methyltransferase, Sss1, as described previously (4,26,27,41). The manner in which this assay is performed produces a reciprocal relationship between the endogenous DNA methylation status and the exogenous [3H]methyl incorporation. All analyses were done in duplicate.
Sodium bisulfite-sequencing assay for colonic p53 methylation
Purified colonic DNA samples from folate-deficient, control and supplemented rats from weeks 1 through to 5 (n = 1/group per each time point) were selected as representatives of their respective groups by choosing that rat whose plasma and colonic mucosal folate concentrations were closest to the respective means of its group. The methylation status of individual CpG sites within the promoter region (between nt -514 and -92) and exons 67 of the p53 gene was determined by the sodium bisulfite-sequencing assay as described previously (23,29) with minor modifications. This method is based on the fact that treatment of denatured DNA with sodium bisulfite converts all cytosine residues to uracil, which are then amplified as thymines in the PCR reactions (42). In contrast, 5-methylcytosine is resistant to bisulfite deamination under the reaction conditions and is amplified as cytosine (42). Sequencing of bisulfite-modified DNA thus allows the positive identification of all methylated cytosine residues within a defined gene sequence (42). Briefly, 2 µg of colonic mucosal DNA was digested with BamH1 (Roche, Laval, Quebec, Canada) followed by denaturation with 0.3 M NaOH for 5 min at 95°C. Freshly prepared sodium bisulfite/urea (Sigma-Aldrich, Oakville, Ontario, Canada) and hydroquinone (Sigma-Aldrich) were added to the denatured DNA at a final concentration of 4.0 M/6.24 M and 0.5 mM, respectively. The addition of urea greatly enhances the reaction efficiency by maintaining the target DNA in single stranded from, thereby allowing complete and reliable conversion (43). The mixture was overlaid with mineral oil and incubated at 55°C for 16 h in the dark. Unreacted bisulfite was removed using the Promega Wizard DNA clean-up desalting column (Promega, Madison, WI). Alkaline desulphonation to uracil was accomplished by the addition of NaOH at a final concentration of 0.3 M with incubation at 37°C for 15 min. The mixture was then neutralized by the addition of ammonium acetate, pH 7.0, to a final concentration of 3.0 M. After ethanol precipitation, the DNA was resuspended in 10 mM TrisHCl/0.1 mM EDTA, pH 8.0 and stored at -20°C. The bisulfite-treated DNA (200 ng) was amplified by PCR in a total reaction volume of 100 µl containing 350 ng of each primer, 0.25 mM each deoxynucleotide triphosphates, PCR buffer (Life Technologies, Gaithersburg, MD), 1.5 mM MgCl2, and 2 U of Platinum Taq DNA polymerase (Life Technologies). The PCR amplification was performed in a thermal cycler (PTC-200 DNA Engine®; MJ Research, Watertown, MA) with the following reaction conditions: 35 cycles of denaturation at 95°C for 20 s, annealing at 54°C for 20 s and extension at 72°C for 30 s. All PCR amplifications included a 10 min extension at 72°C after cycle 35. The product from the first PCR reaction was re-amplified by PCR using nested primers under the same conditions. The primers for the top strand of the promoter region and exons 67 of the rat p53 gene for the first PCR amplification were specifically designed to amplify the sodium bisulfite-modified template based on the published sequence (GeneBank accession nos L07781, L07907 and L07908) (44) according to the recommendations of Clark and Frommer (42) and synthesized by ACGT (Toronto, Ontario, Canada). The sequences of the first set of primers were: promoter region: 5'-TTAAAAAGATGATTATGATTATTTAGTTGG-3' (sense) and 5'-CCAATCTTCAAAAAAACGTAACACCCTAC-3' (antisense); exons 67: 5'-GTTGATTTTTGATTTTTTTTTTTTTTTTATAG-3' (sense) and 5'-ATACCAACCCAACCTAACACACAACTTCC-3' (antisense). The sequences of the nested primers for the second PCR reaction, which contain flanking sequences of EcoRI and XhoI restriction sites to facilitate subcloning into a vector, was constructed based on the published sequence (GeneBank accession nos L07781, L07907 and L07908) (44) and synthesized by ACGT. The sequences of the nested primers were: promoter region: 5'-TTTAGTTGGATAGGAAAGAG-3' (sense) and 5'-CGTAACACCCTACTAAAAAA-3' (antisense); exons 67: 5'-ACACTCGAATTCTTAATAAGTTGTTTTGTTAG-3' (sense) and 5'-CTCACACTCGAGCTAAAATCTTCCAACATAATAATAATAAAA-3'(antisense). The PCR products from the second PCR reaction were gel purified using the Qiaex II Agarose Gel Extraction Kit (Qiagen, Mississauga, Ontario, Canada) according to the manufacturers protocol, re-extracted and dissolved in 50 µl of double-distilled H2O. The PCR products were subcloned into pBluescript II KS(+) vector (Stratagene, Cambridge, UK) at EcoRI and XhoI sites. Over 100 subclones were screened for each sample and 2040 positives were sequenced using the Dideoxy Terminator Label Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and an Applied Biosystems 373 sequencer (Applied Biosystems) as previously described (45,46) to yield the final percent methylation results. In all reactions, the bisulfite-mediated deamination of non-methylated cytosines to uracil was >95% efficient and that methylated cytosine remained >95% resistant to deamination under these conditions.
Steady-state p53 transcripts
Total RNA from the colonic mucosa was isolated by the method of Chomczynski and Sacchi (47). Steady-state levels of the p53 gene among the three groups of rats fed three different levels of dietary folate at each time point were compared by RTPCR followed by Southern hybridization with an internal oligonucleotide probe as described previously (33). Transcripts for the rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene spanning a 618 bp region served as control as described previously (33). The intensity of the hybridized bands was quantified by PhosphoImager using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). p53/GAPDH transcript ratios were calculated and compared among the three dietary groups at each time point. The results are expressed as a percentage of the control.
Statistical analyses
The distribution of each variable was assessed graphically to determine whether it was normally distributed. For normally distributed variables (body weight, folate and homocysteine concentrations), differences among controls and folate-deficient and -supplemented rats were determined by one-way analysis of variance at each time point. For variables that were not normally distributed (SAM and SAH levels, genomic DNA methylation and steady-state levels of p53 mRNA), non-parametric one-way analysis of variance (KruskalWallis test) was used to test differences among the three groups at each time point. Fishers least-significance-difference test and MannWhitney test were used for pairwise comparisons among the different dietary groups for normal and non-normally distributed variables, respectively. The test of linear trend was also performed to assess a trend in changes in values over the study period. Regression analysis was performed to assess correlation between variables. All significance tests were two-tailed, and the significance level was set at 0.05. Results are expressed as mean ± SEM. Statistical analyses were performed by using SYSTAT 5 for Macintosh (Systat, Evanston, IL).
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Results |
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Hematologic indices
Consistent with previous observations (30), hemoglobin levels in the folate-deficient group were 12.4, 12.3 and 36.3% lower than the corresponding values in control and folate-supplemented groups at weeks 3 (P < 0.01), 4 (P < 0.005) and 5 (P < 0.001), respectively (data not shown). In contrast, no differences in hemoglobin levels were observed between the control and folate-supplemented groups at each time point. Mean corpuscular volume was not significantly different among the three groups at each time point (data not shown).
Plasma and colonic mucosal folate and plasma homocysteine concentrations
The mean plasma folate concentrations were significantly different among the three groups beginning with the first week of dietary intervention (P < 0.001; Table I). The mean plasma folate concentrations progressively decreased during the study period in the folate-deficient group (P < 0.001, linear trend), whereas those of the control and folate-supplemented groups did not significantly change over time (Table I
).
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Plasma concentration of homocysteine, which is known to increase in the setting of folate deficiency and is considered to be a more sensitive indicator of cellular folate depletion than blood folate levels (10), increased by 2.5-fold in the folate-deficient group compared with the mean value of control and folate-supplemented groups within the first week of dietary intervention and was 22-fold greater by week 5 (P < 0.01; Table I). In contrast, the mean plasma homocysteine concentrations between the control and folate-supplemented groups were not significantly different at any time point.
Consistent with prior observations in humans and rats (4,48,49), colonic mucosal folate concentrations correlated directly with plasma folate concentrations (r = 0.620.88, P < 0.02) and correlated inversely with plasma homocysteine concentrations (r = -0.600.85, P < 0.02) at each time point. As expected, plasma folate and homocysteine concentrations correlated inversely at each time point (r = -0.700.78, P < 0.005). Overall, including all five time points, colonic mucosal folate concentrations correlated directly with plasma folate concentrations (r = 0.76, P < 0.0001) and correlated inversely with plasma homocysteine concentrations (r = -0.55, P < 0.0001).
Colonic mucosal SAM and SAH concentrations and the ratio of SAM to SAH
The mean concentrations of colonic mucosal SAM, which is the proximal methyl group donor for most biological methylation reactions (10), were not significantly different among the three groups at any time point (Table II). The mean concentrations of colonic mucosal SAH, an accurate inverse indicator of DNA methylation (50,51), were not significantly different among the three groups until week 4 of dietary intervention (Table II
). However, at week 5 of dietary intervention, the mean concentration of colonic mucosal SAH in the folate-deficient group was 3.03.3-fold higher than those in the control and folate-supplemented groups (P < 0.007; Table II
). No significant difference in SAH concentrations was observed, however, between the control and folate-supplemented groups at week 5 (Table II
). The mean colonic mucosal SAM to SAH ratios progressively decreased over the study period (P < 0.001, linear trend), whereas those of the control and folate-supplemented groups did not significantly change during the study period (Table II
). The mean ratio of SAM to SAH was not significantly different among the three groups until week 4 of dietary intervention and by week 5 of dietary intervention, the ratio of SAM to SAH in the folate-deficient group was 6471% lower than those in the control and folate-supplemented groups (P < 0.005; Table II
). No significant difference in the ratio of SAM to SAH was observed between the control and folate-supplemented groups at week 5 (Table II
).
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Colonic p53 methylation
All 15 CpG sites within the promoter region were methylated at a 70100% level at each time point and no significant difference among the three dietary groups was observed at each CpG site at each time point. The extent of methylation at the 10 CpG sites in exons 67 was variable between weeks 1 and 3 and no associations with time or dietary folate were observed (Figure 2A). Over the period extending from weeks 1 to 3, all 10 sites were methylated at an 80100% level (Figure 2A
). At week 4, sites 1 and 2 were slightly hypomethylated compared with other sites at this time point and with the same sites at weeks 13 in both the folate-deficient and control animals (Figure 2B
). At week 4, a 1015% lower level of methylation was observed in the control animal compared with the folate-deficient animal at sites 1 and 2 while at sites 5 and 8, the folate-deficient animal had a 2025% lower level of methylation compared with the control (Figure 2B
). At the end of the experiment (week 5) when folate depletion was at its most severe, the folate-deficient animal had a 65% lower level of methylation at site 1 compared with the control animal (19 versus 84%) whereas the degree of methylation at other CpG sites was not significantly different between the folate-deficient and control animals (Figure 2C
).
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Discussion |
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Although isolated folate deficiency progressively decreased colonic mucosal SAM to SAH ratio during 5 weeks of dietary intervention, only in the extreme deficient state, associated with 20% growth retardation and a 22-fold rise in plasma homocysteine concentration (i.e. week 5), was there a significantly elevated level of colonic mucosal SAH and a significantly reduced colonic mucosal SAM to SAH ratio compared with the control diet. Folate supplementation at four times the basal requirement did not modulate colonic mucosal concentrations of SAM and SAH, and SAM to SAH ratios at any time point. The data are consistent with previous observations made in the setting of a milder, and more chronic state of folate deficiency (4,26), and indicate that modulation of SAM and SAH in the colonic mucosa is particularly resistant to the level of dietary folate. In contrast, folate deficiency, even at a lesser degree than that employed in the present study, has been shown to modulate SAM and SAH in the brain (52), kidney (52), pancreas (53) and liver (2527,34,40) in rats. The tenacious resistance to altered SAM and SAH levels in this tissue compared with others raises the speculation of whether compensatory mechanisms are available due to its exposure to the luminal content of the colon. In this regard, folate that has been synthesized de novo by intestinal microflora is shown to be taken up into colonic epithelial cells by the colonic folate carrier (5456) with subsequent incorporation into host tissues (35).
Plasma and colonic folate concentrations correlated inversely with colonic mucosal SAH concentrations and directly with SAM to SAH ratios, albeit to a modest degree, irrespective of dietary levels of folate. It appears that plasma homocysteine concentrations may be the best predictor of colonic mucosal SAM levels and SAM to SAH ratios. This is entirely consistent with the fact that SAH hydrolase is a reversible reaction that thermodynamically favors the reverse direction (10), thereby causing accumulation of SAH when homocysteine levels are high (Figure 1). Nevertheless, the extent of colonic genomic DNA methylation was not significantly correlated with colonic mucosal SAH levels and SAM to SAH ratios. This is a surprising finding considering that SAH is a potent inhibitor of most SAM-dependent methyltransferases including DNA methyltransferase and that increased plasma and intracellular SAH levels have recently been shown to be an accurate predictor of genomic DNA hypomethylation (50,51). One explanation for the lack of correlation between colonic SAH and genomic DNA methylation is that the range of changes in colonic mucosal SAH levels induced by dietary folate levels employed in the present study is not sufficient to modulate colonic genomic DNA methylation. Another explanation is that a possible compensatory up regulation of DNA methyltransferase might have offset the inhibitory effect of increased SAH associated with folate deficiency. In this regard, combined methyl deficiency has been shown to up regulate DNA methyltransferase activity in rat liver (18,19,23,24).
Interestingly, despite marked folate depletion in plasma and the colonic mucosa and a significant rise in plasma homocysteine concentrations, colonic genomic DNA methylation was significantly higher (by 30%) in the folate-deficient rats than in the controls at week 3. This finding may appear paradoxical but is consistent with prior observations made in rodent liver. For example, a lesser degree of folate deficiency without growth retardation or anemia for 5 weeks in mice induced a significant 56% increase in the degree of genomic DNA methylation in the liver (P < 0.05) followed by the return of genomic DNA methylation value to that of the baseline by 8 weeks (6). These transient increases in genomic methylation observed by us and others may be due to the fact that states associated with diminished availability of methyl group donors result in an enhancement of DNA methyltransferase activity, the enzyme responsible for DNA methylation (18,19,23,24). Therefore, a compensatory up regulation of DNA methyltransferase may transiently increase the extent of genomic DNA methylation in response to folate deficiency until methyl group availability becomes so compromised that the effect is overwhelmed.
The effect of isolated folate deficiency on DNA methylation in the colorectum, the primary target tissue that is particularly susceptible to the folate deficiency-associated carcinogenic effect, has not yet been clearly established. A moderate degree of folate deficiency for 1524 weeks failed to induce significant genomic and c-myc-specific DNA hypomethylation in rat colon (26). The same degree of moderate folate deficiency for 20 weeks in conjunction with an alkylating colon carcinogen, dimethylhydrazine (DMH), did not cause significant genomic DNA hypomethylation in rat colon (4). However, these studies were limited by the fact that the degree of folate deficiency utilized in these studies failed to significantly alter colonic SAM or SAH concentrations (4,26) as well as by the use of DMH (4), which can alter tissue SAM and SAH levels (57) and the extent of DNA methylation (28) independent of the effect of folate. Another recent study showed that a moderate degree of isolated folate deficiency and a combined methyl donor deficiency for 10 weeks, which was associated with DNA strand breaks, did not induce significant genomic DNA hypomethylation in rat colon (59). Taken together, these observations and the data from the present study suggest that isolated folate deficiency does not induce significant genomic DNA hypomethylation in rat colon. Even in rat liver, SAM and SAH concentrations of which are readily modulated by dietary folate levels, the effect of isolated folate deficiency on genomic DNA methylation is not consistent with studies showing hypomethylation (25), no change (26) or hypermethylation (6,27).
There nevertheless are some observations in humans suggesting that altered folate status can affect genomic DNA methylation. Folate depletion in healthy human volunteers in a metabolic unit setting has been observed to diminish genomic DNA methylation in circulating lymphocytes (60,61). In contrast, no significant correlations between genomic lymphocyte DNA methylation and red blood cell folate and plasma homocysteine concentrations were observed in human subjects with normal folate status (62). In some human intervention studies, folate supplementation at 12.525 times the daily requirement significantly increased the extent of colonic genomic DNA methylation (6365) wheras no such effect was observed in lymphocytes with folate supplementation at five times the daily requirement (62). In one human study, serum and cervical tissue folate concentrations correlated inversely, albeit weakly, with cervical genomic DNA methylation (66). However, this study was confounded by the measurement of folate concentrations and genomic DNA methylation in pre-malignant and malignant cervical tissues instead of normal cervical tissue alone (66). However, the data from these human studies collectively raise a possibility that the effect of folate status on genomic DNA methylation may be site and tissue-specific and may depend on the degree of folate depletion and supplementation.
In the present study, the effect of isolated folate deficiency on colonic p53 methylation in the promoter region and exons 67 was variable and not consistent at each CpG site during the course of the experiment and no clear associations with time or dietary folate were observed. The only marked change that occurred specific to the folate-deplete state was hypomethylation at the CpG site 1 at the 5 week time point, and since this change was not evident at any earlier time point, its significance is questionable. This is in marked contrast with prior observations. In one study, the same degree of isolated folate deficiency as in the present study induced significant p53 hypomethylation in exons 67, but not in exon 8, in rat liver (27). Significant p53 hypomethylation in exon 8 was observed in the DMH-treated rat colon in conjunction with folate deficiency, although it remains unclear whether this was due to the DMH, the folate deficiency, or the combination of the two, and this was effectively overcome in a dose-dependent manner by increasing levels of dietary folate (41). Taken together, these observations suggest that isolated folate deficiency does not induce consistent and predictable changes in p53 methylation in rat colon whereas it may produce p53 hypomethylation in specific exons in rat liver and in rat colon in conjunction with alkylating agents. In contrast, dietary depletion of combined methyl donors predictably induces p53 hypomethylation within exons 67 of the p53 gene in rat liver (2224). These observations suggest that p53 methylation changes likely depend on the degree of methyl donor supply and consequent levels of methylation intermediates that are predictably and consistently achieved by combined methyl deficiency and not by isolated folate deficiency. It has been well recognized that cancers from different organs and histologically different subtypes of cancer within a given organ exhibit distinct global and gene-specific methylation patterns (15,67). This suggests that changes in genomic and site-specific DNA methylation patterns in response to folate deficiency may be target organ- and gene-specific, an observation supported by the data from the present study and by prior observations as described earlier.
Although our data indicate that steady-state levels of colonic p53 mRNA correlated directly with plasma and colonic folate concentrations and inversely with plasma homocysteine and colonic SAH levels, no significant correlations were observed with either genomic colonic DNA methylation or colonic p53 methylation in the promoter and exons 67. In contrast, in the methyl deficiency rat model of hepatocarcinogenesis, the degree of p53 methylation within exons 67 was shown to be reciprocally related to the steady-state level of p53 mRNA (24). Our data suggest that changes in steady-state levels of colonic p53 transcript associated with dietary folate deficiency and supplementation are not likely mediated by colonic p53 methylation changes in the promoter or exons 67. In this regard, we have shown previously that steady-state levels of colonic p53 mRNA correlate directly with the integrity of the colonic p53 gene in exons 58 that are readily modulated by folate status in rats (33).
In summary, the data from the present study in conjunction with prior observations (4,6,26,27,41) suggest that isolated folate deficiency is not associated with a significant degree of genomic or p53 DNA hypomethylation in rat colon. This implies that neither genomic hypomethylation nor hypomethylation within the promoter region and exons 67 of the p53 gene is probably a mechanism by which folate deficiency enhances colorectal carcinogenesis in rat models of colorectal cancer. Our data are in marked contrast with the current dogma, which suggests that the folate deficiency-enhanced colorectal carcinogenesis is mediated by genomic and gene-specific DNA hypomethylation. However, we cannot rule out the possibility that a more severe and prolonged extent of folate deficiency with more profound SAM and SAH changes in the colon may modulate genomic and gene-specific DNA methylation, although this degree of folate deficiency is probably not physiologically and clinically relevant in human diseases including colorectal carcinogenesis. Furthermore, our data do not exclude the possibility that sequence-specific alterations of DNA methylation in other portions of the p53 gene, or other cancer-related genes, might be mechanistically involved in colorectal carcinogenesis in the rat. In this regard, a recent in vitro study has demonstrated that human nasopharyngeal carcinoma KB cells grown in folate-deplete medium is associated with hypermethylation in a 5' CpG island and consequent downregulation of the H-cadherin gene compared with cells grown in folate-replete medium (68).
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Notes |
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Acknowledgments |
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References |
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