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,3
* Department of Molecular and Cellular Biochemistry, ** Comprehensive Cancer Center, and Department of Internal Medicine, Ohio State University, Columbus, OH 43210 and Division of Biochemical Toxicology, Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079
3 To whom correspondence should be addressed. E-mail: jacob.42{at}osu.edu or ghoshal.1{at}osu.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • hepatocarcinogenesis • folate- and methyl-deficient diet • Dnmt1/3a/3b • MBD1–4
Epidemiological studies indicate that inadequate dietary intake of folate may predispose humans to increased cancer risk [for review, see (1)]. Many micronutrients and vitamins are indispensable for metabolic pathways (2,3). Folate, an important mediator in the transfer of methyl groups, is essential for the de novo biosynthesis of purines and thymidylate and plays a crucial role in DNA synthesis, stability, and integrity. Aberrations in any or all of these processes have been implicated in colorectal (4,5) and hepatocellular carcinogenesis (6,7). Folic acid is metabolized to 5-methyltetrahydrofolate that acts as a methyl donor in the conversion of homocysteine to L-methionine. L-Methionine is subsequently metabolized to S-adenosyl-L-methionine (SAM)4, the principal methyl donor in the majority of biochemical reactions, including methylation of macromolecules. Folate deficiency causes depletion of SAM, resulting in genome-wide DNA hypomethylation, activation of oncogenes, and malignant transformation (1). Paradoxically, recent studies show that limited folate intake also results in hypermethylation of the CpG islands of many tumor suppressor genes (4,8).
Methylation at 5-position of cytosine is the predominant epigenetic modification in the mammalian genome that is essential for development [for review, see (9,10)]. It is heritable due to the activity of DNA methyltransferase (Dnmt) (see Fig. 1A). DNA methylation is initiated by de novo DNA methyltransferases Dnmt3a and Dnmt3b, which is propagated in the newly replicated DNA strand by the maintenance DNA methyltransferase, Dnmt1 (11,12). The C-terminal catalytic domains of these enzymes contain motifs that are homologous to bacterial methyltransferase. Their N-terminal regions harbor several domains that interact with DNA and protein. The major function of DNA methylation is the silencing of retroviral promoters, transposable elements in the genome, and regulating expression of imprinted genes (13). Apart from methyltransferase activity, these enzymes also act as transcriptional repressors by recruiting corepressors like histone deacetylase and histone methyltransferase (Fig. 1B) (14,15).
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Previous studies in rodents have shown that a diet deficient in methionine and devoid of folic acid and choline (FMD diet) induces preneoplastic nodules in the livers of male F344 rats after wk 36 and hepatocellular carcinomas after wk 54 (7,17). Recent studies have shown that this diet induces global hypomethylation specifically in the liver, the target tissue susceptible to neoplastic transformation (17). This diet causes the depletion of SAM and a decrease in the SAM/S-adenosyl homocysteine (SAH) ratio, leading to genome-wide hypomethylation (18). Interestingly, this diet also induces gene-specific hypermethylation (7). We used restriction landmark genomic scanning (RLGS) analysis to demonstrate genome-wide alteration in the methylation profile in preneoplastic nodules and carcinomas (19). One of the hypermethylated genes was identified as the receptor-type protein, tyrosine phosphatase (PTPRO), which was subsequently found to be methylated in different types of human cancer, including those of the liver, lung (20), and colon (21).
To elucidate the molecular mechanisms by which the FMD diet induces de novo methylation and the silencing of specific genes, we made a systematic analysis of the expression profile of the components of DNA methylation machinery that include Dnmts and MBDs at early stages of hepatocarcinogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Measurement of SAM and SAH levels. SAM and SAH in the liver were measured by HPLC, as described previously (23).
RNA isolation and reverse transcription. Frozen livers were pulverized and used for RNA, DNA, and nuclei isolation. Total RNA was isolated with the guanidinium isothiocyanate-acid phenol method, as described earlier (24). Reverse transcription was carried out with random hexamers and M-MuLV reverse transcriptase from 3 µg of total RNA in 20 µL of total volume, following the protocol provided in the GeneAmp RNA PCR kit (Applied Biosystems).
Real time RT-PCR analysis. An aliquot of the cDNA (equivalent to 100 ng of RNA for Dnmt1, 250 ng for each of Dnmt3A or 3B, and 10 ng for 18S rRNA) was used for real-time RT-PCR analysis as described (15,25). The optimum primer concentration was 150 nmol/L. A standard curve for each cDNA was first generated using 10-fold serial dilutions (108–102 copies) of the respective cDNAs as a template. To create the standard curve, rat 18S rRNA, Dnmt1, Dnmt3a, and Dnmt3b, MBD1–4, and MeCP2 cDNAs were amplified by RT-PCR. The copy number of each cDNA expressed in rat liver was calculated from the standard curve and normalized to that of 18S rRNA. The primer sequences and conditions for RT-PCR are described in Supplemental Table S3.
Isolation of tissue nuclei and preparation of nuclear extract. The nuclei were isolated from individual rat livers by homogenization in hypotonic buffer containing 2 mol/L sucrose, which was followed by sucrose density gradient centrifugation, as described (26,27). The nuclear pellet from each sample was resuspended in the lysis buffer (50 mmol/L Tris-HCl, pH 8.10; 1 mmol/L EDTA, pH 8; and 10 g/L SDS) containing a protease inhibitor cocktail (Sigma Chemical) and was followed by brief sonication to shear viscous DNA. The supernatant obtained after centrifuging the extract at 20,000 x g for 10 min was subjected to Western blot analysis with anti-Dnmt1, Dnmt3a, Dnmt3b, MBD1–4, and MeCP2 antibodies, using protocols described earlier (24,27,28). Antibodies against all MBDs, Dnmt3a, and Dnmt3b were raised in our laboratory (24,27,28). Antibodies against Dnmt3a and 3b were raised against their N-terminal domains that lack a conserved catalytic site (27). MBDs were raised against the recombinant protein fragments that lacked a highly conserved methyl CpG binding domain at the N-terminus. Antibody against Dnmt1 was a generous gift from Dr. Shoji Tajima (29). ß-Tubulin antibody was from Santa Cruz Biotechnology. An aliquot of nuclear extract (200 µg for Dnmts and MBD1, 2, and 4, and 50 µg for MBD3) was used for immunoblot analysis.
Statistical analysis.
The data were analyzed by a 2-way ANOVA (diet x age) using the SPSS statistical package (SPSS, version 13.0). When the assumption of homogeneity of variances was violated with Levene's test showing a P 
0.01, we log transformed the data prior to analysis. Due to multiple comparisons and resulting 
-inflation, overall main effects and interaction P-value were required to be 0.001 to be considered significant, whereas pairwise tests were required to have P < 0.01. If overall P-values were 0.001 for treatment and age, pairwise t tests with adjusted were used to differentiate between control and FMD treatments at each age. Linear correlation was performed using Pearson correlation with P 0.01 considered significant. Values presented are means ± SD.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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RNA, a marker for preneoplastic transformation of hepatocytes, as early as wk 9; this level was maintained until wk 36 (Table 1). Similarly, the hepatic SAM concentration, a marker for methionine level, was reduced to 50–60% (P < 0.002) of controls in rats fed the FMD diet at wk 9, wk 18, and wk 36 without significant changes in hepatic SAH (data not shown) (18). These results confirmed that these rats were indeed methyl deficient, and preneoplastic changes in hepatocytes occurred in all rats fed the FMD diet.
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The hepatic Dnmt1 protein level was also elevated (P < 0.001) in rats fed the FMD diet (Fig. 2, a representative Western blot; and Table 2). Although Dnmt1 protein was not affected by age and the interaction was not significant, the diet-induced increase was 2.8-fold at wk 9, 1-fold at wk 18, and 1.2-fold at wk 36. The correlation between RNA and protein levels (r = 0.60, P < 0.01) indicates that folate and methyl deficiency induces Dnmt1 gene expression at a transcriptional or posttranscriptional level by stabilizing its mRNA. The FMD diet-induced increase in hepatic Dnmt3a mRNA level was 1.8-, 2.1-, and 1-fold at wk 9, wk 18, and wk 36, respectively (Table 1), whereas its protein level was not affected by diet or age (Table 2). In contrast, the Dnmt3b RNA and protein levels were not affected by diet or age (Tables 1 and 2). These results demonstrate that upregulation of both de novo (Dnmt3a) and maintenance (Dnmt1) methyltransferases is an early event in FMD diet–induced hepatocarcinogenesis.
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0.001) in the livers of rats fed the FMD diet than in controls (Table 3). The increases in hepatic MBD1 mRNA were comparable at wk 9 (70%), wk 18 (120%), and wk 36 (100%). The FMD diet induced a 2.2-fold increase in hepatic MBD2 mRNA at wk 9, which was greater than those at wk 18 (1.8-fold) and wk 36 (1.4-fold). The upregulation in MBD3 mRNA was maximum (150%) at wk 9, after which it declined at wk 18 and wk 36 to 120% (Table 3). The expression of MBD2 and MBD3, like that of Dnmt1, decreased (P < 0.001) with age. The MBD2 mRNA level was highest at wk 9, after which it decreased with age to 66 and 47% of the wk 9 level at wk 18 and wk 36, respectively (Table 3). In the FMD diet-fed rats, the MBD2 mRNA level declined to 58 and 35% of the wk 9 level at wk 18 and wk 36, respectively. In the controls, the decrease in MBD3 level was 29% at wk 18 followed by a 43% decrease at wk 36. The decreases were more robust (by 65 and 71% at wk 18 and wk 36, respectively) in the FMD fed rats. Neither diet nor age affected the mRNA level of MeCP2 and MBD4.
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1-, 3-, and 2.4-fold greater at wk 9, wk 18, and wk 36, respectively (Table 4). Among the MBDs, MeCP2 protein was unique because its level increased with age in both groups compared with the wk 9 controls (P < 0.001). Among the controls, MeCP2 protein increased 4.4- and 9-fold at wk 18 and wk 36, respectively, compared with wk 9 (Table 4). In the deficient group, the increase in MeCP2 level was comparable at wk 18 (90%) and wk 36 (100%). Because MBD4 and MeCP2 mRNA levels were not affected by age (Table 3), these data suggest that the age-dependent increase in these proteins is probably due to translational or posttranslational mechanisms.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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50 CpG among 1500 islands analyzed by RLGS (19). As a first step to elucidate the underlying mechanism, we studied the effect of this dietary regimen on the expression of proteins involved in DNA methylation-mediated gene silencing. To our knowledge, this is the first comprehensive study of the alteration in the DNA methylation machinery at early stages of tumor development. The mRNA and protein levels of some Dnmts and MBDs in the liver were altered in response to methyl deficiency for 36 wk, when preneoplastic nodules became visible. Significant upregulation of specific Dnmts and MBDs at early stages of this nutritional deficiency suggests their potential role in the neoplastic process. Knock-out or hypomorphic mice will be useful in addressing the functional implication of the differential expression of some of these factors in the carcinogenic process. Global hypomethylation, particularly of the repetitive elements and oncogenes, is a common epigenetic event during early stages of carcinogenesis (30,31). The intriguing observation is that, in addition to DNA hypomethylation, folate and methyl deficiency can lead to localized or regional hypermethylation and suppression of a few genes that include tumor suppressor genes (4,19). This finding is consistent with a profound and progressive increase in the transcript and protein levels of Dnmt1 at the early stage of hepatocarcinogenesis. Interestingly, the mRNA levels of all 3 Dnmts were upregulated in human hepatocellular carcinoma (8,32,33). However, in those studies, the protein levels of these enzymes were not measured. Recent study has shown that global DNA hypomethylation in rats fed the FMD diet occurs specifically in livers that undergo neoplastic changes (17). The limiting SAM concentration in the deficient liver is probably responsible for global DNA hypomethylation.
Analysis of the transcript and protein levels of the MBDs revealed many striking features. In agreement with the previous observation (34), the mRNA and protein levels of MBD2 were upregulated in the methyl-deficient liver. Upregulation of MBD2 expression in response to methyl deficiency is of particular importance in view of similar observations made in other experimental systems. Thus, MBD2 has been shown to play a role in the methylation-mediated suppression of human ribosomal RNA (35) and mouse metallothionein-I genes (36). Unlike the promoters of tumor suppressor genes transcribed by RNA polymerase II, the ribosomal gene, transcribed by RNA polymerase I, was hypomethylated in human hepatocellular carcinomas relative to matching liver tissue (35). In another example, MBD2 was markedly upregulated in benign breast tumors compared with normal breast tissues, whereas there was no significant change in MeCP2 expression in the breast tumor (37).
The expression of 2 other members, namely, MBD1 and MBD4, followed a pattern similar to that of Dnmt1, exhibiting an increased expression of folate and methyl deficiency at wk 9. This observation indicates that there are changes in the expression of these components of epigenetic regulation at the early stages of hepatocyte transformation. Elevated expression of MBDs and the associated corepressor complex with the methylated promoter are likely involved in the methylation-mediated silencing of p16 (38) and PTPRO (the gene encoding the receptor-type protein tyrosine phosphatase-type O) (19) in the livers of rats fed the FMD diet.
Earlier studies reveal DNA damage in exon 5 of the p53 gene in the rat liver following 9 wk of folate deficiency (22). Folate deficiency causes uracil misincorporation into DNA, which, in turn, leads to single and double strand breakages in DNA. MBD4, the methyl CpG binding protein, is likely to be responsible for DNA mismatch repair. MBD4 exhibits strong DNA glycosylase activity and preference for G:T mismatches (39) that probably occurs as a result of deamination of 5-methyl cytosines in a symmetrically methylated CpG base pair. Moreover, MBD4 also functions as transcriptional repressor for methylated DNA (40). Upregulation of MBD4 protein in the livers of folate-deficient rats probably facilitates the elimination of CpG:TpG mismatches in DNA caused by methyl deficiency.
It is noteworthy that the mammalian Dnmts can act as transcriptional repressors because of their relatively large N-terminal regions, which are absent in bacterial methyltransferasess (Fig. 1) (41,42). We have recently shown that differentiation of PC12 (pheochromocytoma) cells to neuronal cells results in upregulation of Dnmt3b and inhibition of Dnmt3b expression by RNA interference and leads to continuous proliferation of the cells and failure to generate neurites (15). The striking observation was that Dnmt3b-mediated neuronal differentiation occurred in the absence of its catalytic activity. It is possible that Dnmt1 and Dnmt3a act in concert initially to repress target gene promoters that are subsequently hypermethylated and permanently silenced. Additional experiments are needed to test this hypothesis.
Switching rats to normal diets after consuming the FMD diet can replete the hepatic SAM level and the SAM/SAH ratio only in those that had consumed the FMD diet for 9 wk, but not for 18 wk or 36 wk (18). The inability to recover the hepatic SAM level and the DNA methylation profile in rats consuming the diet for 18 wk or 36 wk before shifting them to a folate- and methyl-adequate diet suggests that irreversible changes had occurred in the livers undergoing preneoplastic transformation. The inability to reverse mRNA profiles of Dnmts and MBDs toward normal levels (data not shown) further supports the notion that prolonged dietary deficiency induces permanent genetic and epigenetic changes in the liver that cannot be reversed by consuming a normal diet at a later stage.
In conclusion, the present study shows that substantial alterations in the expression of DNA methyltransferases and methyl CpG binding proteins occur at early stages of hepatocarciniogenesis induced by folate and methyl deficiency in rats. These changes may be one of the important contributing factors in the formation of epigenetically reprogrammed cells that become neoplastic.
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FOOTNOTES |
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2 Supplemental Tables 1–3 are available with the online posting of this paper at www.nutrition.org.
4 Abbreviations used: Dnmt, DNA methyltransferase; FMD diet, folate- and methyl-deficient diet; MBD, methyl CpG binding domain protein; PTPRO, receptor type protein tyrosine phosphatase; RLGS, restriction landmark genomic scanning; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine.
Manuscript received 15 December 2005. Initial review completed 3 January 2006. Revision accepted 23 February 2006.
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