Impact of Dnmt1 deficiency, with and without low folate diets, on tumor numbers and DNA methylation in Min mice
Jacquetta Trasler1,2,
Liyuan Deng1,
Stepan Melnyk3,
Igor Pogribny3,
Francois Hiou-Tim1,
Sahar Sibani1,
Christopher Oakes1,2,
En Li4,
S. Jill James3 and
Rima Rozen1,5
1 Departments of Pediatrics and Human Genetics, McGill University-Montreal Children's Hospital, 4060 Ste. Catherine St. West, Montreal, Quebec H3Z 2Z3, Canada,
2 Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada,
3 Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arizona, USA and
4 Massachussetts General Hospital, Charlestown and Harvard Medical School, Boston, Massachussetts, USA
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Abstract
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Although a number of studies have suggested that diets with low intake of folate, an important methyl donor, are associated with increased risks of colon cancer and its precursor the adenomatous polyp, the underlying mechanisms are poorly understood. Dysregulation and instability of DNA methylation and alterations in the levels of the predominant DNA methylating enzyme, DNA (cytosine-5)-methyltransferase 1 (Dnmt1), have also been linked to tumorigenesis. We have used a combination of genetic and dietary manipulation to assess the effects of reduced Dnmt1 expression with and without folate deficiency on tumor induction in the ApcMin mouse. ApcMin mice with a reduction in Dnmt1 expression (ApcMin/+/Dnmt1C/+) had significantly lower tumor numbers than ApcMin mice with normal Dnmt1 (ApcMin/+/Dnmt1+/+). Dietary folate deficiency from weaning to 13 weeks of age did not affect tumor number or size in ApcMin/+/Dnmt+/+ mice. However, in ApcMin/+/Dnmt1C/+ mice with high baseline tumor numbers (41 ± 4), folate deficiency was associated with a decreased absolute number of tumors (27 ± 3), but a higher proportion of larger tumors as compared with mice on the control diet. In the repeat experiment, ApcMin/+/Dnmt1C/+ mice had low baseline tumor numbers (20 ± 2) and folate deficiency did not affect tumor number (23 ± 4) or size as compared with the same mice on the control diet. These results suggest that, in the presence of Dnmt1 deficiency, the effects of folate deficiency on tumor number and size may depend on the stage of adenoma development when folate deficiency is initiated. We also show that folate deficiency with or without reductions in Dnmt1 did not affect overall genomic DNA methylation or the methylation levels of two candidate genes, E-cadherin or p53, in normal or neoplastic intestinal tissue. In conclusion, genetic deficiency in Dnmt1 with or without folate deficiency decreases tumor number in the ApcMin mouse model, but this effect may not be mediated by changes in SAM or SAH levels, nor by alterations in global methylation in the pre-neoplastic intestinal tissue.
Abbreviations: Dnmt1, DNA (cytosine-5)-methyltransferase 1; SAM, S-adenosylmethionine
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Introduction
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A number of human epidemiological studies have suggested that diets with a low intake of the methyl donors folate and methionine are associated with increased risks of colon cancer, and of its precursor the adenomatous polyp (17). However, the mechanisms underlying the inverse relationship between dietary folate intake and the risk of colorectal adenomas and cancer are poorly understood. Critical folate-dependent reactions relevant for tumorigenesis include methionine synthesis and nucleotide synthesis. 5-Methyltetrahydrofolate (5-methyl THF) is the predominant plasma form of folate and is in limited supply in folate deficiency. 5-Methyl THF provides the methyl group for methionine and S-adenosylmethionine (SAM), the methyl donor for DNA methylation as well as for numerous other biological reactions.
It has been postulated that low folate status may increase the risk of colon cancer through alterations in DNA methylation. Methylation of cytosine residues in DNA occurs for the most part within CpG dinucleotides and is a post-replication process catalyzed by DNA methyltransferases (Dnmts) (reviewed in ref. 8). Methylation of CpG sites within the promoter region of genes is associated with transcriptional repression (9,10). Both global DNA hypomethylation as well as regional hypermethylation have been found in a number of cancers (11). In rat studies, DNA from livers of animals fed folate-/methyl-deficient diets has been reported to be hypomethylated (1214). DNA hypomethylation has also been found in lymphocytes of humans on low dietary folate and can be reversed by folate repletion (15). In support of a connection between folate/methyl deficiency and regional alterations in DNA methylation, deficiency of methyl groups in rats was associated with p53 hypomethylation during the first 36 weeks of deficiency followed by p53 hypermethylation after 54 weeks (16).
Sites of methylation in DNA are also mutagenic `hotspots', as 5-methylcytosine spontaneously deaminates to thymine giving rise to C to T transition mutations. DNA methylation changes associated with cancer include mutations at CpG sites in the tumor suppressor genes p53 and p16, alterations in overall DNA methylation and high levels of Dnmt activity (17). Hypermethylation of the promoter of E-cadherin, an invasion suppressor gene, has been reported in several different carcinoma cell lines (18). Folate deficiency could contribute to DNA methylation errors and increased mutation rates by reducing levels of SAM. When SAM is limiting, Dnmt1 can promote the deamination of cytosine to uracil and subsequent C
T transition mutations. Thus, the high rates of mutation seen at CpG sites may be due to both spontaneous deamination of 5-methylcytosine as well as enzymatic deamination of cytosine when methyl group supply is low (19).
Mouse models have contributed to our understanding of the etiology of intestinal cancer. One well-studied model of intestinal neoplasia is the Min (or ApcMin) mouse that has a heterozygous germ-line mutation at codon 850 of the mouse Apc gene and develops 2575 small intestinal adenomas and one to five colorectal adenomas by 160180 days (20,21). The human homolog of Apc, APC, is mutated in a majority of sporadic human colorectal cancers (22).
The effect of folate intake has been examined on tumor numbers in ApcMin mice, which also have a knockout of the mismatch repair gene, Msh2 (ApcMin/+Msh2-/- mice) (23). MSH2 is altered in 21% of families with hereditary non-polyposis colorectal cancer (24). In this mouse model, folate supplementation led to a reduction in tumor numbers if administered prior to tumor development; in contrast, folate deficiency conferred protection after tumor formation (23). These folate-supplemented and deficient diets were assessed by the same group for their ability to alter tumor numbers in the ApcMin/+ (Min) mouse (25). Significant changes in tumor numbers throughout the whole small intestine were not observed, although Song et al. (25) identified a significant linear decrease in the number of ileal adenomas with increasing dietary folate intake. In our earlier studies in Min mice, we found that tumor numbers correlated with SAM levels, SAH levels and with global DNA hypomethylation in the pre-neoplastic mucosa of mice fed standard lab chow (26). We also tested the effects of folate-deficient diets on tumor number. We concluded from three separate experiments that the modulatory role of folate might correlate with the transformation state of the cell (26).
Effects of reduced Dnmt1 levels in Min mice have been studied by crossing Min mice with mice heterozygous for two different Dnmt1 targeted mutations (Dnmt1S/+ and Dnmt1N/+) (27,28). Dnmt1-deficient Min mice showed a dramatic reduction in tumor numbers compared with Min mice with wild-type Dnmt1 levels (27,28). The hypomethylating drug 5-azacytidine caused a further reduction in tumor numbers in the ApcMin+/- DnmtS/+ mice (27). Detailed DNA methylation studies were not carried out in the folate-deficiency studies (23,25) nor in the Dnmt1-deficiency studies (27,28) of Min mice. Recently, complete suppression of polyp formation was reported in Min mice with a combination of two different Dnmt1 mutations (29).
To determine whether methylation changes contribute to the variation in tumor numbers in the Min mouse, we elected to use a combination of genetic (Dnmt1 haplo-insufficiency) and dietary manipulation (folate deficiency) to assess the impact on global methylation and on tumor number/size in Min mice. We also evaluated the methylation profile of two candidate genes, E-cadherin and p53.
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Materials and methods
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Animals and diets
ApcMin/+ mice on a C57BL/6 background were purchased from Jackson Laboratories (Bar Harbor, MA) and genotyped using a PCR-based assay (23). DnmtC/+ mice, also on a C57BL/6 background and heterozygous for a mutation in the catalytic domain of Dnmt1, have been described previously (30). A PCR assay was designed to genotype the DnmtC/+ mice using primers exon33S (5'-AGCTACTGTGACTACTACCGGC-3') and exon33As (5'-ACCTGGAGCACACCAAAGGTGC-3') to amplify a 146 bp fragment in Dnmt1 exon 33 which is only present on wild-type alleles and primer intron31S (5'-GTGGTGCGATGCATGTTTGAGCA-3') and pgkAs (5'-AAGTGCCAGCGGGGCTGCTAAA-3') to amplify an 81 bp fragment specific for the targeted allele. ApcMin/+ male mice were crossed with DnmtC/+ females. Offspring of each of the resulting genotypes received either a control or a folate-deficient diet from weaning until 13 weeks of age. The control/folate-deficient diets were synthesized in the laboratory with the following constituents (units for each are grams/kilogram of diet where for each constituent the first number is that for the control diet and the second number is that for the folate-deficient diet): casein, 100/100; soy protein, 100/100; soybean oil, 70/70; cellulose, 47.5/47.5; cornstarch, 170/170; sucrose, 450/450; mineral mix, 45/45; folate-free vitamin mix, 12/12; choline, 3/1.5; BHT, 0.014/0.014; folate, 0.002/0; L-cystine, 3.3/3.3. The amount of folate in the control diet is the recommended value for rodents (2 mg/kg diet) whereas the folate-deficient diet had no added folic acid. However, we cannot exclude the possibility that trace levels of folate were present in some of the other dietary constituents such as casein; this would induce a moderate rather than a severe folate deficiency. The same dietary experiment was performed twice.
Polyp analysis
The mice were killed at 13 weeks of age and the intestines were removed. Some of the tumors and sections of non-involved intestine were removed from each mouse, snap frozen and stored at 70°C, for the biochemical measurements; the tumor number in these sections was determined before freezing. The remaining intestine was fixed in Bouin's solution and fatty tissue on the mucosal surface was removed. Tumor number and size were determined from the fixed tissue using a dissecting microscope with a micrometer; this was performed independently by two different individuals blinded to the genotypes of the mice.
Biochemical measurements and DNA methylation analysis
Intracellular SAM and SAH levels were measured by an HPLC-based method (31). Global DNA methylation was ascertained using three different assays, the cytosine extension assay (32), a thin-layer chromatography assay (33) and the methyl acceptance assay (34). Briefly, for the cytosine extension assay, high molecular weight DNA was digested with the methylation-sensitive restriction enzyme HpaII and a single nucleotide extension reaction was performed with 0.5 U AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA) and 0.1 µl of [3H]dCTP (57.4 Ci/mmol, NEN, Boston, MA) as described previously in detail (32). In this assay, the incorporation of [3H]dCTP is directly proportional to the number of unmethylated (cleaved) CpG sites in the original sample, and thus reflects the level of global DNA hypomethylation. A well-characterized thin-layer chromatography assay that assesses the methylation status of CCGG sites was performed exactly as described previously (33) and quantified by phosphorimagery. The third assay for assessment of global DNA methylation was the methyl acceptance assay in which the bacterial enzyme SssI transfers radiolabeled methyl groups to unmethylated CpG sites in DNA. In this assay, radiolabel incorporation is inversely proportional to the number of unmethylated CpG sites (34).
Bisulfite genomic sequencing (35) was used to examine site-specific methylation in the promoter region of E-cadherin (from 555 to +29 relative to the initiation codon, GenBank accession number M81449) and exons 58 of p53 (position 199336200818, accession number ACO74146.1 in HTGS division of GenBank). For E-cadherin, two fragments were amplified using PCR primers for the first fragment coding strand (sense: 5'-GGAAGAAGAGAATTGATTT TTGAAGGTTG-3'; antisense: 5'-CTTCCTCCACCCCTATCTATAATTAATAAC-3') and non-coding strand (sense: 5'-GAGAAATAGTTTAGTTAGTAAAGGTTAATG-3'; antisense: 5'-CACCTACAAATAACAACCAAAAAACTAC-3') and for the second fragment coding strand (sense: 5'-TGGGTTAGAGTATAGTTAGGTTAGGATT-3'; antisense: 5'-AACCATAAAAAAACCTACAACAAAAACAAA-3') and non-coding strand (sense: 5'-ATGATTAAAGTTTTTTGTAATTTTATGTTT-3'; antisense: 5'-ACTACAAAACTCAAACTCCAACTCC-3'). Two fragments were amplified for p53 using PCR primers for the first fragment coding strand (sense: 5'-GGTGTTTAATGGTGTTTGGATAATGTG-3'; antisense: 5'-CATCAATCTAAACTAAAATCAACTATCTC-3') and non-coding strand (sense: 5'-GGTGTTTAATGGTGTTTGGATAATGTG-3'; antisense: 5'-ACACCTCTAAACCTAACTAACACTC-3') and for the second fragment coding strand (sense: 5'-GGTTATTTGTAGTGAGGTAGGGAG-3'; antisense: 5'-TTAAAATAAAACTCAACAAACTCCTCC-3') and non-coding strand (sense: 5'-ATTTTTGGTTGTTTAGGTTATTTGTAG-3'; antisense: 5'-TTAAAATAAAACTCAACAAACTCCTCC-3'). PCR products were subcloned using the TOPO TA Cloning kit (Invitrogen). Plasmid DNA was isolated with the QIAprep Spin Miniprep Kit (Qiagen, Mississanga, Canada) and sequenced on an Applied Biosystems (Foster City, USA) automated sequencer.
Direct sequencing of PCR products for mutation analysis of exons 58 of p53 was carried out to check for mutations associated with folate- and/or Dnmt1-deficiency in the ApcMin/+ mice. Primers were designed to amplify each of the four exons as follows: exon 5 (sense: 5'-TTGTCCCCGACCTCCGTTCTCTCTC-3'; antisense: 5'-GAGATGGGAGGCT GCCAGTCCTAA-3'); exon 6 (sense: 5'-CCTCCCATCTCCCGGCTTCTGACTT-3'; antisense: 5'-GAGGGTGAGGCAAACGGGTTGCTA-3'); exon 7 (sense: 5'-AGGTAGGGAGCGACTTCACCTGGA-3'; antisense: 5'-CCAAAGAGCGTTGGGCATGTGGTA-3'); exon 8 (sense: 5'-TCAGGATGGGGCCCA-GCTTTCTTC-3'; antisense: 5'-TGGGGTGAAGCTCAACAGGCTCCT-3').
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Results
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DnmtC/+ decreases the size and number of tumors in ApcMin/+ mice
Previous studies have reported that two single targeted mutations of Dnmt1, DnmtS/+ and DnmtN/+, can decrease tumor numbers in ApcMin/+ mice (27,28). Here, in both experiment 1 (Figure 1A
, control diet) and experiment 2 (Figure 2A
, control diet), we show that a third single Dnmt1 mutation, in DnmtC/+ mice, is also associated with a decreased number of tumors in ApcMin/+ mice. In addition, in both experiments, there was a significantly increased percentage of larger tumors in mice with wild-type Dnmt1 levels (ApcMin/+/Dnmt1+/+) as compared with mice with reduced levels of Dnmt1 (ApcMin/+/DnmtC/+) (Figures 1B and 2B
).

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Fig. 1. Effect of Dnmt1 deficiency with and without folate deficiency on tumor number (A) and size distribution (B) in ApcMin mice in experiment 1. (A) Number of tumors is shown (± SEM). The number of mice examined for each genotype is shown beneath each vertical bar. (B) The size distribution (percentage, total value 1.0) of tumors in each category is shown (± SEM). *P 0.05, ANOVA, compared with the C/+ group on the control diet.
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Fig. 2. Effect of Dnmt1 deficiency with and without folate deficiency on tumor numbers (A) and size distribution (B) in ApcMin mice in experiment 2. (A) Number of tumors (± SEM) is shown. The number of mice in each group is indicated beneath each vertical bar. (B) The size distribution (percentage, total value 1.0) of the tumors (± SEM) in each category is shown. *P < 0.05, ANOVA, compared with the C/+ group with the same diet.
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Effects of dietary folate deficiency
Both low dietary folate ingestion and DNA methylation abnormalities have been associated with an increased risk of colorectal cancer. We combined dietary and genetic manipulation to assess the effects of reduced Dnmt1 levels and folate deficiency on ApcMin-induced tumorigenesis in the mouse. In the first experiment (Figure 1
) ApcMin/+/Dnmt1C/+ mice on a control diet had an average of 41 ± 4 tumors. In this experiment, folate deficiency in the ApcMin/+/DnmtC/+ mice was associated with a decreased absolute number of tumors (27 ± 3, Figure 1A
) but a higher proportion of larger tumors (Figure 1B
).
In the second experiment (Figure 2
), the ApcMin/+/DnmtC/+ mice on the control diet had an average of only 20 ± 2 tumors (Figure 2A
), ~50% the number found for mice of the same genotype in the first experiment (Figure 1A
). Neither tumor number (23 ± 4, Figure 2A
) nor size distribution (Figure 2B
) were affected by 10 weeks of folate-deficient diet in the second experiment.
Based on clinical evidence supporting an inverse relationship between folate status and risk for colorectal tumors (17), we asked whether, in our mouse model, low levels of dietary folate could enhance or accelerate tumorigenesis in ApcMin mice. This was tested in the second experiment in which tumor numbers and size in ApcMin/+/Dnmt+/+ mice on a control diet were compared with those ApcMin/+/Dnmt+/+ mice on a folate-deficient diet. Mean tumor number in mice on the folate-deficient diet (54 ± 6) was lower but not statistically different from those on the control diet (62 ± 6, Figure 2A
); in addition, the distribution of tumor sizes was nearly identical on the two different diets (Figure 2B
). Thus, in our model, 10 weeks of folate deficiency does not appear to accelerate tumorigenesis in ApcMin/+ mice.
SAM, SAH and global DNA methylation in normal and neoplastic intestinal tissue
Dietary folate is an important source of methyl groups for a number of biochemical processes, including the production of SAM, a universal methyl donor also needed for cytosine methylation in DNA. The effect of the control and folate-deficient diets on SAM and SAH levels in the normal (non-adenomatous) intestine is shown in Table I
. Folate deficiency resulted in a significant decrease in SAM levels in almost all groups. In the group in which the decreased SAM was not statistically significant, a significant increase in SAH was observed. The observed alterations in SAM or SAH are characteristic of folate deficiency and indicate the effectiveness of the folate-deficient diet (36,37).
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Table I. SAM and SAH concentrations, and DNA methylation levels in preneoplastic small intestine. DNA hypomethylation results were obtained by the cytosine extension assay; DNA methylation (%methylated CCGG) results were obtained by the thin layer chromatography assay, as described in Methods section. All values represent mean ± S.E.M. for 5-6 samples except for the DNA methylation (% methylated CCGG) results where n = 3-6 samples. * = P<0.05 by Student t test.
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It is postulated that lower levels of SAM can result in DNA hypomethylation. However, in our experiments, none of the mice on folate-deficient diets showed changes in DNA methylation of pre-neoplastic small intestinal tissue (Table I
). Similarly, Min mice heterozygous for a mutation in the catalytic domain of Dnmt1 might also be expected to have decreased global DNA methylation in the small intestine, compared with Min mice without the Dnmt mutation. In fact, DNA methylation levels were not statistically different in Min mice with and without the Dnmt mutation, using the dCTP incorporation and the TLC assays (Table I
). A third assay (SssI methyl acceptance assay) also did not reveal any differences in DNA methylation between these two groups (data not shown). There was a small but statistically significant decrease in global methylation using the TLC assay in the Dnmt mutant mice, compared with wild-type Dnmt mice, in the absence of the Min mutation. However, as we did not see any differences in methylation when the Min mutation was present in conjunction with the Dnmt1 mutant allele, we conclude that substantial global DNA hypomethylation does not appear to be associated with the decreased tumor numbers present in the ApcMin/+/DnmtC/+ mice.
Site-specific methylation of E-cadherin
The promoter region of E-cadherin is unmethylated in most cells and epigenetic silencing by promoter hypermethylation has been reported in a number of cancers including colon cancer (17). We used bisulfite sequencing to evaluate the methylation status of 41 CpG sites within the E-cadherin promoter region in the pre-neoplastic and neoplastic intestinal tissue of ApcMin/+/DnmtC/+ mice on control and folate-deficient diets (Figure 3
). There was no evidence of hypermethylation at any individual site or across sites in the intestinal tissues of mice on the control or folate-deficient diets.

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Fig. 3. Effects of folate-deficient diets on the methylation of the promoter region of E-cadherin in the pre-neoplastic and neoplastic small intestine of ApcMin/+/Dnmt1C/+ mice. Individual clones are shown where the number in front of each clone indicates the number of times that particular methylation pattern was seen. Open circles indicate an unmethylated site. Filled circles indicate a methylated site.
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Site-specific methylation and mutation analysis of p53
The region of p53 stretching between exons 58 contains a relatively high number of CpG dinucleotides that are predominantly methylated in normal cells. Alterations in methylation in this region have been reported in rats on folate-deficient diets (16). To assess the possibility of regional areas of hypomethylation in a normally methylated gene, we used bisulfite sequencing to evaluate the methylation status of 45 CpG sites in the exon 58 region of p53. Results for the ApcMin/+/DnmtC/+ and ApcMin/+/Dnmt+/+ mice on the control diet, animals that exhibit large differences in tumor number (Figure 2A
), are shown in Figure 4
. For both genotypes, there was a similar level of methylation in the pre-neoplastic and tumor DNA, indicating that, in our model, Dnmt1 deficiency was not associated with hypomethylation of p53. In addition, folate deficiency with or without Dnmt1 deficiency did not affect p53 methylation or result in widespread hypomethylation across exons 58 (data not shown).

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Fig. 4. Effects of Dnmt1 deficiency on the methylation of exons 58 of p53 in pre-neoplastic and neoplastic small intestinal tissue. Individual clones are shown where open circles indicate unmethylated sites and filled circles indicate methylated sites.
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An alternative DNA methylation alteration associated with cancer includes mutations in tumor suppressor genes such as p53. Direct sequence of p53 exons 58 in mice of all genotypes (n = 3 mice/genotype) on normal and folate-deficient diets did not reveal any mutations (data not shown).
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Discussion
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An inverse relationship between dietary folate intake and the risk of colon cancer has been reported in a number of studies. One postulated mechanism by which folate deficiency may contribute to tumorigenesis is through alterations in DNA methylation. Genomic and regional alterations in DNA methylation and alterations in the levels of DNA methyltransferases have been linked to tumorigenesis (17,19). In this report we show that both Dnmt1 deficiency and folate deficiency can modulate tumor numbers in Min mice without causing a concomitant alteration in overall genomic DNA methylation levels, suggesting that other mechanisms are involved.
We showed that Min mice heterozygous for a targeted mutation in the catalytic domain of Dnmt1 (Dnmt1C/+) had a much lower tumor number and size than Min mice with wild-type levels of Dnmt1. Effects on tumor number are consistent with previous results in Min mice heterozygous for two other single mutations in Dnmt1, Dnmt1S/+ (27) and Dnmt1N/+ (28,29) mice. In addition, as in our study, the Min/DnmtN/+ mice also showed a decrease in tumor size as compared with the Min/Dnmt+/+ mice (28,29). The precise mechanism underlying the decrease in tumor size and numbers in Dnmt1-deficient Min mice is unclear. As the divergence in tumor size and number between the mice deficient in Dnmt1 compared with those with wild-type Dnmt1 levels appeared to occur after 70 days of age (28), the authors suggested that gene promoter hypermethylation may lead to silencing of a gene that negatively regulated tumor growth; Dnmt1 deficiency might then decrease the likelihood of de novo promoter methylation. Support for this hypothesis comes from experiments performed in the ApcMin/DnmtS/+ mice where the hypomethylating drug 5'-aza-2'-deoxycytidine was able to further decrease tumor numbers (27). In previous studies of Dnmt1-deficient Min mice, methylation of centromeric repeats was examined using a Southern blotting assay, but significant differences between wild-type and Dnmt-deficient Min mice were not observed (27,29). Here we used three separate assays to assess global methylation and did not observe methylation differences in Min mice with and without a Dnmt mutation. Therefore, all studies thus far have failed to identify major changes in global DNA methylation as a contributing factor to decreased tumor numbers in this experimental model.
Folate deficiency is potentially an alternate way to decrease DNA methylation levels. In our experiments, folate deficiency combined with Dnmt1 deficiency was associated with a decreased tumor number in the first but not the second experiment, even though the protocols did not vary between the two experiments. However, in the first experiment, ApcMin/+Dnmt1C/+ mice on the control diet for 10 weeks had 2-fold higher baseline tumor numbers than mice of the same genotype in the second experiment. The variation in baseline tumor numbers in this mouse model is well known, with a reported range of 2575 adenomas (20,21). We suggest that the timing of initiation of folate-deficient diets in relation to the stage of adenoma development may be critical; i.e. folate deficiency initiated after the establishment of adenomas, or later in their developmental program, as in experiment 1, can lead to a decrease in tumor numbers whereas when folate deficiency is initiated before the establishment of adenomas (experiment 2), folate deficiency has little effect. A similar observation was made by Song et al. (23) where a moderately folate-deficient diet started after the establishment of neoplastic foci significantly reduced the number of intestinal adenomas in ApcMin/Msh2-/- mice. Our results are also consistent with previous studies in our laboratory, in which the administration of the same folate-deficient diet as in this study resulted in decreased tumor numbers in Min mice when the baseline tumor numbers were high (26).
In the two experiments in this study, the tumor numbers were quite similar in Min mice on the control diet that were wild-type for Dnmt (68 and 62 tumors, Figures 1A and 2A
). In the first experiment, Dnmt deficiency decreased tumor numbers by ~40%, to 41 tumors; the addition of folate deficiency further reduced tumor numbers down to 27 tumors. In the second experiment, the decrease in tumor numbers due to Dnmt deficiency was more dramatic, a decrease of 70%, to 20 tumors. It is possible that folate deficiency contributed to an additional decrease in tumor numbers in the first experiment, down to 27 tumors, as the decrease due to Dnmt deficiency alone was not as extensive as that in the second experiment. Interestingly, in the dietary experiments with Min mice in our previous study (26), the lowest tumor numbers that we obtained were also approximately 20. Thus, there may be a maximum tumor number reduction that is achievable with a single Dnmt mutation or with a folate-deficient diet in mice with the germ-line APC mutation.
Despite decreases in SAM, folate deficiency did not lead to an alteration in overall genomic methylation levels in the intestine of the Dnmt+/+ mice. In addition, alterations in small intestinal DNA methylation were not seen in mice deficient in Dnmt1 (Dnmt C/+) with or without the folate-deficient diet. The fact that the folate-deficient diet used in the current experiments was effective is evidenced by either decreased SAM levels or increased SAH levels. Interestingly, however, neither SAH nor SAM levels differed significantly between the ApcMin mice that were deficient in Dnmt as compared with those that had wild-type Dnmt1 levels.
We also looked for regional alterations in DNA methylation. We chose two genes that have been implicated in colon carcinogenesis, one that is normally unmethylated, E-cadherin, and one that is normally methylated, p53 (17). Both candidate genes showed normal methylation patterns in ApcMin mice that were Dnmt1 deficient with or without folate deficiency. Although we cannot rule out other site-specific methylation changes, the results on E-cadherin and p53 are consistent with the lack of an effect on global DNA methylation. In a recent study on Min mice (29), Dnmt1 deficiency was associated with a decrease in DNA methylation of a small number of genes; however, the involvement of these genes in tumorigenesis is unclear. Interestingly, p16, a tumor suppressor gene like E-cadherin and p53 that has been implicated in carcinogenesis, was not affected (29). Thus, to date we do not know of any clear tumorigenesis-associated genes whose methylation status is altered in a way that would explain the decreased tumor numbers in the Dnmt1-deficient Min mice. Results from the Eads et al. (38) study where a few genes were affected suggest that more sensitive genome-wide scanning techniques such as restriction landmark genomic scanning may be required to reveal gene- and individual animal-specific DNA methylation alterations in critical genes.
In conclusion, both Dnmt1 deficiency and folate deficiency can reduce tumor number and size in the small intestines of Min mice. Our studies did not provide support for the hypothesis that Dnmt or folate-deficiency modulate tumor number through effects on DNA methylation. Although more exhaustive DNA methylation studies, such as the use of microarrays and restriction landmark genomic scanning are still warranted, other mechanisms of Dnmt1- and folate-dependent tumorigenesis, such as induction of mutations and DNA damage, should also be actively pursued.
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Notes
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5 To whom correspondence should be addressed Email: marlene.aardse{at}mcgill.ca 
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Acknowledgments
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This work was supported by the Cancer Research Society of Canada. J.T. is a Scientist of the Canadian Institutes for Health Research (CIHR) and a Scholar of the Fonds de la recherché en santé du Québec. R.R. is a Senior Scientist of the CIHR.
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Received July 23, 2002;
revised September 18, 2002;
accepted September 22, 2002.