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U.S. Department of Agriculture, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202-9034
2To whom correspondence should be addressed. E-mail: cdavis{at}gfhnrc.ars.usda.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • selenium • DNA methylation • azadeoxycytidine • HT-29 • DNA methyltransferase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). It is estimated that half of the Western population can expect to develop at least one colorectal tumor by age 70 (2
). Supplemental selenium has been found to reduce the incidence and mortality of colon cancer in humans (3
). This is consistent with animal studies showing protective effects of selenium against aberrant crypt formation and colon tumor development (4
–7
). Aberrant colonic crypts and aberrant crypt foci are preneoplastic lesions in the colon that are statistically associated with the number of tumors that ultimately develop. As such, they are excellent biomarkers for determining colon cancer risk without going through a lengthy tumor study.
Genetic alterations are a hallmark of human cancer. In addition to these genetic alterations, changes in DNA methylation, an epigenetic modification present in mammalian cells, are another hallmark of human cancer (8
). Progressive dysregulation and disruption of the heritable patterns of DNA methylation have been a consistent observation during multistage carcinogenesis. During tumor progression, the DNA becomes paradoxically hypomethylated, despite the presence of regional hypermethylation and an increase in DNA methyltransferase (Dnmt1)3
activity (9
,10
). Methylation changes the interactions between proteins and DNA, which leads to alterations in chromatin structure and either a decrease or an increase in the rate of transcription (11
). Methylation of a promoter CpG island leads to binding of methylated CpG binding proteins and transcriptional repressors, including histone deacetylases, and to a block of transcription initiation (12
,13
). It has been proposed that dietary factors such as folate, alcohol and methionine may be associated with colon cancer because of their involvement in DNA methylation processes (14
). We recently observed that rats fed selenium-deficient diets had significantly hypomethylated liver and colon DNA compared with rats fed diets supplemented with selenite or selenomethionine (15
). Thus, alteration in DNA methylation may be a potential mechanism whereby deficient dietary selenium increases liver and colon tumorigenesis.
Increased Dnmt1 activity has also been shown to stimulate colon tumorigenesis (16
). In in vitro model systems, overexpression of Dnmt1 has been shown to trigger transformation (17
,18
) and to be an essential molecular step in c-fos–mediated transformation (19
). Dnmt1 activity may also be essential for transformation in vivo; Min mice carrying defective Apc genes at high risk for developing colonic adenomas develop fewer polyps when carrying disrupted alleles for Dnmt1, a gene encoding a DNA methyltransferase likely capable of both maintenance and de novo CpG dinucleotide methylation, than when carrying normal Dnmt1 alleles (20
,21
).
The compound 5-aza-2'-deoxycytidine (aza-dC) inhibits DNA methylation and often is used in vitro to induce the reexpression of genes putatively silenced by promoter methylation (22
). Aza-dC is substituted for cytosine during replication and is recognized by Dnmt1 (23
). Attempted transfer of methyl groups to aza-dC, however, covalently traps the enzyme to newly synthesized DNA (24
,25
). This sequestration ultimately depletes cellular stores of Dnmt1 and results in widespread genomic hypomethylation (24
,25
). The purpose of the current studies was to determine whether aza-dC treatment would affect the inhibition of aberrant crypt formation observed with dietary selenium in rats. Because the colons were utilized in the rat study for aberrant crypt analysis and were not available for analysis of DNA methylation or Dnmt1 activity, a second objective was to determine the effect of dietary selenium and aza-dC treatment on global DNA methylation and Dnmt1 protein expression in HT-29 cells, a human colon adenocarcinoma cell line.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Weanling male Fischer-344 rats (n = 60) were purchased from Sasco (Omaha, NE). All rats were housed individually in stainless steel wire-bottomed cages in a room with controlled temperature and light. Rats were provided free access to demineralized water and purified diet. The basal diet was a selenium-deficient, torula yeast–based diet. The basal diet contained 300 g/kg torula yeast, 3 g/kg DL-methionine, 590 g/kg sucrose, 50 g/kg corn oil, 35 g/kg selenium-deficient AIN-76A mineral mix (26
), 12 g/kg calcium carbonate, 10 g/kg AIN-76A vitamin mix, 0.1 g/kg choline bitartrate and 0.01 g/kg menadione sodium bisulfite complex. The basal diet was supplemented with 0, 0.1 or 2 mg selenium/kg diet as sodium selenite (by analysis, the diets contained 0.004, 0.11 and 2.03 mg selenium/kg diet, respectively). Half of the rats were injected weekly with 5-aza-2'-deoxycytidine (1 mg/kg, subcutaneously) and half were injected with the vehicle control (PBS) for the entire 12 wk of the study. After 3 wk of consuming the experimental diets, the rats were given two injections, separated by 1 wk, of dimethylhydrazine (DMH; 25 mg/kg, intraperitoneally). Rats were maintained on their diets for an additional 8 wk.
This study was approved by the Animal Care Committee of the Grand Forks Human Nutrition Research Center, and the rats were maintained in accordance with the guidelines for the care and use of laboratory animals.
Sample collection.
Food was withheld overnight before rats were anesthetized with xylazine (Rompon, Moboay, Shawnee, KS) and ketamine (Ketaset, Aveco, Fort Dodge, IA) and killed by exsanguination. Blood was collected by cardiac puncture into syringes containing EDTA such that the final concentration was 
1 g EDTA/L blood. The colon and rectum were removed, flushed with 9 g/L NaCl, opened longitudinally and fixed flat between paper towels in 700 mL/L ethanol. The colon and rectum were stored in 700 mL/L ethanol at 4°C before analysis.
Analysis of aberrant crypt foci.
The fixed colon and rectum were stained with 1 g/L methylene blue in 0.1 mol/L sodium phosphate buffer (pH 7.4). Aberrant crypt foci and the total number of aberrant crypts were scored without knowledge of their source by using a dissecting microscope to visualize the aberrant crypt foci as previously described (4
).
Selenium status.
Selenium concentrations in the plasma, liver and colon were determined by hydride-generation atomic absorption spectrometry according to a published procedure (27
). Samples were prepared for analysis by predigestion in nitric acid and hydrogen peroxide, followed by high temperature ashing while in the presence of MgNO3 as an aid to prevent Se volatilization.
Glutathione peroxidase enzyme activity was determined by the coupled enzymatic method of Paglia and Valentine (28
), which uses hydrogen peroxide as the substrate.
Liver S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH).
Portions of fresh liver were weighed and homogenized at 11,500 rpm in 0.4 mol/L HClO4 by using a Mark II Tissumizer (Tekmar, Cincinnati, OH). Samples were centrifuged at 2000 x g at 4°C for 30 min. The supernatant was stored at -70°C until analysis. SAM and SAH were measured on a Dionex 40000i HPLC (Dionex, Sunnyvale, CA) according to the procedure of Bottliglieri (29
).
Plasma homocysteine and cysteine.
Total homocysteine and cysteine were determined in heparinized plasma by using HPLC according to the procedure of Durand et al. (30
).
Cell culture.
HT-29 cells were obtained from American Type Culture Collection (Rockville, MD). Cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco BRL, Rockville, MD) with 100 mL/L fetal bovine serum, 0.1 mmol/L nonessential amino acids and 1 mmol/L sodium pyruvate in a humidified incubator at 37°C with an atmosphere of 5% CO2. Sodium selenite and aza-dC were purchased from Sigma Chemical (St Louis, MO). Exposure to selenite and/or aza-dC was made 24 h after cells were seeded onto culture flasks (675,000 cells were plated onto 25 cm2 in 5 mL of medium) to ensure that neither aza-dC nor selenite affected attachment to the plastic substrate. HT-29 cells were exposed to basal medium supplemented with 0, 1 or 2 µmol selenium/L in the presence and absence of 500 nmol aza-dC/L; all determinations were performed in triplicate. The basal medium with 100 mL/L fetal bovine serum contained 0.53 µmol selenium/L. Aza-dC was freshly prepared and filter sterilized just before use. Cells not treated with aza-dC were treated with an equal volume of PBS. After 24 h, the medium was changed and all flasks were exposed to the same concentration of selenium in the absence of aza-dC. Medium containing 0, 1 or 2 µmol selenite/L was changed every 2 d. After 4 additional days of growth in the presence of selenite, flasks containing the cells were lysed with warmed (37°C) 5 g/L SDS in 10 mmol/L Tris, 10 mmol/L EDTA, pH 8.0. DNA was isolated by a procedure involving enzymatic digestion of protein and RNA followed by extraction with phenol and chloroform/isoamyl alcohol (24:1) (31
). DNA concentration was determined spectrophotometrically at 260 nm by using a value of 50 A260 absorbance units/mg DNA to calculate its concentration.
Genomic DNA methylation.
The methylation status of CpG sites in genomic DNA was determined by the in vitro methyl acceptance capacity of DNA by using [3H-methyl]SAM as a methyl donor and a prokaryotic CpG Dnmt1, as described (15
,32
–34
). The manner in which this assay is performed produces a reciprocal relationship between the endogenous DNA methylation status and the exogenous 3H-methyl incorporation. Briefly, 2 µg of DNA was incubated with 185 kBq of [3H-methyl]SAM (Amersham Life Science, Piscataway, NJ), 4 U of Sss1 methyltransferase (New England Biolabs, Beverly, MA), 1 X Sss1 buffer (50 mmol/L, NaCl, 10 mmol/L Tris-HCl, 10 mmol/L EDTA, 1 mmol/L dithiothreitol, pH 8.0) as previously described. All analyses were done in duplicate.
Immumoblot analysis.
Cells were scraped into PBS and were disrupted with a tissue sonicator. The disrupted cells were combined with Laemmli sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 250 mL/L glycerol, 20 g/L SDS, 0.1 g/L bromophenol blue and 350 mmol/L dithiothrietol) and heated at 100°C for 5 min. Samples (75 µg total protein) were electrophoresed on 6.5% polyacrylamide gels in the presence of SDS. After electrophoresis, gels and polyvinylidene fluoride blotting membranes (Immobilin-P, Millipore, Bedford, MA) were equilibrated with transfer buffer (12 mmol/L Tris, 96 mmol/L glycine and 150 mL/L methanol) for 30 min. Gels were transblotted for 5 min at 8 V and 40 min at 26 V. After transfer, nonspecific binding of primary antibody was blocked with 50 g/L nonfat dry milk in Tris buffered saline (TBS; 10 mmol/L Tris, 150 mmol/L NaCl, pH 8.0) overnight. Blocking buffer was decanted and blots were incubated with anti-Dnmt1 antibodies at a concentration of 2 mg/L in 50 g/L nonfat dry milk and 5 mL/L Tween 20 in TBS for 2 h at room temperature. The Dnmt1 antibody used was an affinity-purified immunoglobulin G preparation isolated from rabbit antiserum raised against a polypeptide N-MADANSPPKPLSKPRTPRRS-C (derived from human Dnmt-1) (16
) at Research Genetics (Huntsville, AL). Horseradish peroxidase-conjugated anti-rabbit antibody (Gibco BRL) was used as the secondary antibody. Membranes were incubated for 1 min in a 1:1 solution of ECL Plus Western Blot detection reagents (Amersham Life Science, Arlington Heights, IL) and then exposed to X-ray film. The bands corresponding to the 190-kDa protein were quantified with a densitometer.
Statistical analyses.
The data were analyzed by a two-way ANOVA (diet selenium and treatment) using the SAS general linear models program (SAS Version 6.12, SAS Institute, Cary, NC). Tukey’s contrasts were used to differentiate among means for variables that had been significantly (P < 0.05) affected by selenium or by a selenium x treatment interaction. Because the numbers of aberrant crypts and aberrant crypt foci were assumed to follow a Poisson distribution, the data were analyzed by using a generalized linear model. Bonferroni adjustments were used when comparing means to account for multiple comparisons. Values are reported as means ± SEM in the text. Data with unequal variances were not transformed before statistical analysis because the unequal variances did not affect the results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Both the concentration of selenium and treatment with aza-dC significantly (P < 0.004) influenced the weight gain of the rats (Table 1
). Rats fed the selenium-adequate (0.1 mg Se/kg diet) diet weighed significantly more than rats fed the selenium-deficient or selenium-supplemented diets. Rats injected with aza-dC weighed significantly less than rats injected with PBS.
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). Rats fed the selenium-deficient diet and injected with PBS had significantly (P < 0.006) more aberrant crypts than rats fed 0.1 or 2.0 mg selenium/kg diet (244 ± 21 vs. 165 ± 9 and 132 ± 14, respectively). In contrast, when rats were injected with aza-dC, there was a significant (P < 0.0001) reduction in aberrant crypt formation and dietary selenium had no effect (62 ± 8 vs. 77 ± 13 vs. 54 ± 8, in rats fed 0, 0.1 and 2.0 mg selenium/kg diet, respectively). Similar results were obtained for the total number of aberrant crypt foci (Fig. 1)
.
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. Rats fed the selenium-deficient diet had significantly (P < 0.0001) lower serum, erythrocyte and liver glutathione peroxidase activities and significantly (P < 0.0001) lower colon, plasma and liver selenium concentrations than rats fed adequate dietary selenium. Similarly, rats fed the selenium-adequate diet (0.1 mg Se/kg diet) had significantly (P < 0.0001) lower erythrocyte and liver glutathione peroxidase activities and significantly (P < 0.0001) lower colon, plasma and liver selenium concentrations than rats fed supplemental dietary selenium (2 mg Se/kg diet).
Rats fed the selenium-deficient diets had significantly (P < 0.0001) lower plasma homocysteine and cysteine concentrations compared with rats fed 0.1 or 2.0 mg selenium/kg diet as selenite (Table 2
). Rats injected with aza-dC had 10% lower (P < 0.03) plasma homocysteine concentrations than rats injected with PBS. Neither dietary selenium nor aza-dC treatment significantly affected liver SAM or liver SAH. However, dietary selenium caused a dose-dependent decrease in the ratio of liver SAM/SAH (5.32 vs. 4.87 vs. 4.24 in rats fed 0, 0.1 and 2.0 mg Se/kg diet, respectively).
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By using SssI methylase, the relative content of 5-methyl cytosine was assessed in samples of DNA from HT-29 cells grown in the presence of 0, 1 or 2 µmol selenite/L added and 0 or 500 nmol aza-dC/L. In this assay, the number of methyl groups incorporated into DNA in the presence of 3H-SAM and bacterial SssI methylase is proportional to the original number of CpG sites available for methylation. Thus, it is inversely proportional to the prior methylation status of DNA. The DNA isolated from HT-29 cells not treated with selenite was significantly (P < 0.001) hypomethylated compared with that isolated from cells treated with 1 or 2 µmol selenium/L (Fig. 2
). Cells treated with 1 µmol selenium/L were significantly (P < 0.003) hypomethylated compared with cells treated with 2 µmol selenium/L. Aza-dC treatment resulted in significant (P < 0.006) DNA hypomethylation (75369 vs. 55323 dpm/µg DNA in cells treated with aza-dC vs. PBS, respectively). There were no significant interactive effects of media selenium concentration and aza-dC treatment on DNA methylation.
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). Cells grown in the absence of additional selenium had higher Dnmt1 protein expression than cells grown in the presence of 2 µmol selenium/L. Aza-dC treatment did not affect Dnmt1 protein expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,25
), the lack of an effect of dietary selenium on aberrant crypt formation in rats injected with aza-dC suggests that decreased Dnmt1 activity may protect selenium-deficient rats against carcinogen-induced aberrant crypt formation. However, the data may also indicate that the cellular effects of aza-dC are more important in suppressing aberrant crypts than the increase in aberrant crypts caused by selenium deficiency. Although it is likely that the efficacy of aza-dC relates to methyltransferase activity, it might also relate to induction of methylation-silenced tumor suppressor genes, genomic hypomethylation and/or enzyme-DNA adduct formation. Unfortunately, the present studies do not allow us to determine which is the most important and which is most influenced by selenium status. Because colon tissue was unavailable for further analysis after the quantification of aberrant crypts, we assessed the effect of dietary selenium and aza-dC treatment on global DNA methylation and Dnmt1 protein expression in HT-29 cells, a human colon adenocarcinoma cell line. Aza-dC treatment caused significant hypomethylation of the DNA but did not affect Dnmt1 protein expression. This result is not surprising because we measured protein expression rather than activity. Aza-dC functions to covalently trap the enzyme, not to degrade it. HT-29 cells cultured in the absence of additional media selenium had significantly hypermethylated DNA but less Dnmt1 protein expression than cells cultured in the presence of 1 or 2 µmol selenium/L. The decreased Dnmt1 protein expression in cells cultured in the presence of selenium suggests that selenium can inhibit Dnmt1 protein expression and therefore may inhibit Dnmt1 activity.
Similarly, studies from other investigators have suggested that dietary selenium can inhibit Dnmt1 activity in vitro (35
–37
). Fiala et al. (35
) observed that sodium selenite, benzyl selenocyanate and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibit Dnmt1 extracted from nuclei of a human colonic carcinoma. p-XSC also inhibits the enzyme in HCT116 human carcinoma cells in culture at a concentration of 0–40 µmol/L (35
). Cox and Goorha reported the inhibition of Dnmt1 partially purified from Friend erythroleukemic cells (36
) and rat liver (37
) by sodium selenite in vitro at a range of 1–100 µmol selenium/L. Our studies differ from the previous studies in that we investigated Dnmt1 protein expression in the cells at physiologic concentrations of selenium. In contrast, the previous studies (35
–37
) primarily investigated the effect of selenium on the activity of the purified enzyme in vitro at very high concentrations of selenium. However, these results all suggest that inhibition of Dnmt1 activity by dietary selenium might be a mechanism for its chemopreventive effect.
In the current study, HT-29 cells cultured in the absence of selenium had global DNA hypomethylation despite higher Dnmt1 protein expression. Although these results seem paradoxical because Dnmt1 functions to methylate DNA at cytosine residues, many other investigators have obtained similar results. For example, Fiala et al. (35
) observed increased Dnmt1 activity and global DNA hypomethylation in colon tumor samples compared with adjacent uninvolved mucosa. Consumption of methyl-deficient diets has been shown to decrease DNA methylation and to increase Dnmt1 activity. Slack et al. (38
) offered a molecular explanation for the documented coexistence of global hypomethylation and high levels of Dnmt1 activity in many cancer cells. They demonstrated that an AP-1–dependent regulatory element of Dnmt1 is heavily methylated in most somatic tissues and in the mouse embryonal cell line P19, and completely unmethylated in a mouse adrenal carcinoma cell line, Y1 (38
). Dnmt1 is highly overexpressed in Y1 relative to P19 cell lines. Global inhibition of DNA methylation in P19 cells by aza-dC results in demethylation of the AP-1 regulatory region and induction of Dnmt1 expression in P19 cells, but not in Y1 cells (38
). Thus, they hypothesize that the AP-1 regulatory region of Dnmt1 acts as a sensor of the DNA methylation capacity of the cell (38
).
Dietary selenium may be inhibiting Dnmt1 activity by affecting the AP-1 regulatory region of Dnmt1. Previous investigators have observed that selenite inhibits the binding of the transcription factor AP-1 to DNA via selenite-mediated oxidation of cysteine residues in its leucine zipper region (39
,40
). Future studies are warranted to investigate the effect of dietary selenium on the methylation status and the binding of AP-1 to the AP-1 regulatory region of Dnmt1.
In the current study, both selenium deficiency and aza-dC treatment caused DNA hypomethylation in HT-29 cells. Similarly, in a previous study, we observed that deficient dietary selenium caused global hypomethylation of liver and colon DNA in experimental rats and in Caco-2 cells (15
). However, selenium deficiency increased and aza-dC treatment decreased aberrant crypt formation in rats treated with DMH. These results suggest that changes in DNA methylation do not appear to be the mechanism for the chemopreventive effects of dietary selenium.
Selenium deficiency also altered methyl metabolism in the rats. Rats fed the selenium-deficient diet had a large decrease in plasma homocysteine and cysteine concentrations and an increased ratio of SAM to SAH in the liver. We have also recently observed that rats fed a selenium-deficient diet have significantly decreased betaine homocysteine methyltransferase activity, which is one of the enzymes that synthesizes methionine from homocysteine, and significantly increased plasma total free glutathione concentrations (41
). This suggests that less homocysteine is being remethylated to form methionine and more is being routed through the transulfuration pathway to form glutathione. Perhaps the higher ratio of SAM to SAH reflects the global hypomethylation of the DNA. Dnmt1 catalyzes the transfer of methyl groups from SAM to dC residues in DNA. This produces 5-methylcytosine and SAH. However, it should be noted that rats fed folate-deficient diets have increased plasma homocysteine concentrations and decreased ratios of SAM to SAH despite hypomethylation of DNA (42
).
In conclusion, aza-dC treatment, which inhibits Dnmt1 activity, protects selenium-deficient rats against carcinogen-induced aberrant crypt formation. HT-29 cells cultured in the absence of selenium had significantly more Dnmt1 protein expression than cells cultured in the presence of selenium. Other investigators have suggested that dietary selenium can inhibit Dnmt1 activity. Together, these results suggest that inhibition of Dnmt1 activity by dietary selenium may be a mechanism for its chemopreventive effect. However, future studies must investigate the effect of dietary selenium on cancer susceptibility, global and gene-specific DNA methylation, Dnmt1 activity and DNA demethylase activity at the same time to determine whether the chemopreventive effects of selenium may be due to changes in DNA methylation, DNA demethylase activity and/or Dnmt1 activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: aza-dC, 5-aza-2'-deoxycytidine; DMH, dimethylhydrazine; Dnmt1, DNA methyltransferase; p-XSC1, 4-phenylenebis(methylene)selenocyanate; SAH, S-adenosyl-homocysteine; SAM, S-adenosyl-methionine; TBS, Tris buffered saline.
Manuscript received 20 August 2001. Initial review completed 10 October 2001. Revision accepted 6 November 2001.
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LITERATURE CITED |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. American Cancer Society (2000) Cancer Facts and Figures 2000:4 ACS Atlanta, GA. .
2. Kinzler, K. & Vogelstein, B. (1996) Lessons from hereditary colorectal cancer. Cell 87:159-170.[Medline]
3.
Clark, L. C., Combs, G. F., Turnbull, B. W., Slate, E. H., Chalker, D. K., Chow, J., Davis, L. S., Glovar, R. A., Graham, G. F., Gross, E. G., Krongrad, A., Lesher, J. L., Park, K., Sanders, B. S., Smith, C. L. & Taylor, J. R. (1996) Effect of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. J. Am. Med. Assoc. 276:1957-1963.
4. Feng, Y., Finley, J. W., Davis, C. D., Becker, W. K., Fretland, A. J. & Hein, D. W. (1999) Dietary selenium reduces the formation of aberrant crypts in rats administered 3,2'-dimethyl-4-aminobiphenyl. Toxicol. Appl. Pharmacol. 157:36-42.[Medline]
5. Finley, J. W., Ip, C., Lisk, D. J., Davis, C. D., Hintze, K. J. & Whanger, P. D. (2001) Cancer-protective properties of high-selenium broccoli. J. Agric. Food Chem. 49:2679-2683.[Medline]
6. Jao, S.-W., Shen, K.-L., Lee, W. & Ho, Y.-S. (1996) Effect of selenium on 1,2-dimethylhydrazine-induced intestinal cancer in rats. Dis. Colon Rectum 39:628-631.[Medline]
7. Reddy, B. S., Wynn, T. T., El-Bayoumy, K., Upadhyaya, P., Fiala, E. & Rao, C. V. (1996) Evaluation of organoselenium compounds for potential chemopreventive properties in colon cancer. Anticancer Res 16:1123-1128.[Medline]
8. Herman, J. G. (1999) Hypermethylation of tumor suppressor genes in cancer. Semin. Cancer Biol. 9:359-367.[Medline]
9. Baylin, S. B., Herman, G. G., Graff, J. R., Vertino, P. M. & Issa, J.-P. (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Cancer Res 72:141-196.
10.
Jones, P. A. & Gonzalgo, M. L. (1997) Altered DNA methylation and genome instability: a new pathway to cancer?. Proc. Natl. Acad. Sci. U.S.A. 94:2103-2105.
11.
Jones, P. A. & Takai, D. (2001) The role of DNA methylation in mammalian epigenetics. Science (Washington DC) 293:1068-1070.
12. Jones, P. L., Veenstra, G. J., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., Strouboulis, J. & Wolffe, A. P. (1998) Metylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19:187-191.[Medline]
13. Nan, X., Ng, H. H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenman, R. N. & Bird, A. (1998) Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature (Lond.) 393:386-389.[Medline]
14. Slattery, M. L., Schaffer, D., Edwards, S. L., Ma, K. N. & Potter, J. D. (1997) Are dietary factors involved in DNA methylation associated with colon cancer?. Nutr. Cancer 28:52-62.[Medline]
15.
Davis, C. D., Uthus, E. O. & Finley, J. W. (2000) Dietary selenium and arsenic affect DNA methylation in vitro in Caco-2 cells and in vivo in rat liver and colon. J. Nutr. 130:2903-2909.
16.
DeMarzo, A. M., Marchi, V. L., Yang, E. S., Veeraswamy, R., Lin, X. & Nelson, W. G. (1999) Abnormal regulation of DNA methyltransferase expression during colorectal carcinogenesis. Cancer Res 59:3855-3860.
17.
Wu, J., Issa, J. P., Herman, J., Bassett, D. E., Nelkin, B. D. & Baylin, S. B. (1993) Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells [see comments]. Proc. Natl. Acad. Sci. US.A. 90:8891-8895.
18.
Wu, J., Herman, J. G., Wilson, G., Lee, R. Y., Yen, R. W., Mabry, M., de Bustros, A., Nelkin, B. D. & Baylin, S. B. (1996) Expression of prokaryotic HhaI DNA methyltransferase in transforming and lethal to NIN 3T3 cells. Cancer Res 56:616-622.
19.
Bakin, A. V. & Curran, T. (1999) Role of DNA 5-methylcytosine transferase in cell transformation by fos. Science (Washington, DC) 283:387-390.
20.
Pradham, S., Talbot, D., Sha, M., Benner, J., Hornstra, L., Li, E., Jaenisch, R. & Roberts, R. J. (1997) Baculovirus-mediated expression and characterization of the full-length murine DNA methyltransferase. Nucleic Acids Res 25:4666-4673.
21. Laird, P. W., Jackson-Grusby, L., Fazeli, A., Dickinson, S. L., Jung, W. E., Li, E., Weinberg, R. A. & Jaenisch, R. (1995) Suppression of intestinal neoplasia by DNA hypomethylation. Cell 81:197-205.[Medline]
22. Bender, C. M., Zingg, J. M. & Jones, P. A. (1995) DNA methylation as a target for drug design. Pharm. Res. 15:175-187.
23. Momparler, R. L. (1985) Molecular, cellular and animal pharmacology of 5'-aza-2'-deoxycytidine. Pharmacol. Ther. 30:287-299.[Medline]
24.
Juttermann, R., Li, E. & Jaenisch, R. (1994) Toxicity of 5-aza-2'-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proc. Natl. Acad. Sci. U.S.A. 91:11797-11801.
25.
Ferguson, A. T., Vertino, P. M., Spitzner, J. R., Baylin, S. B., Muller, M. T. & Davidson, N. E. (1997) Role of estrogen receptor gene demethylation and DNA methyltransferase. DNA adduct formation in 5-aza-2`-deoxycytidine-induced cytotoxicity in human breast cancer cells. J. Biol. Chem. 272:32260-32266.
26. American Institute of Nutrition (1977) Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 107:1340-1348.
27. Finley, J., Matthys, L., Schuler, T. & Korynta, E. (1996) Selenium content of foods purchased in North Dakota. Nutr. Res. 16:723-728.
28. Paglia, D. & Valentine, W. (1967) Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70:158-169.[Medline]
29. Bottliglieri, T. (1990) Isocratic high performance liquid chromatographic analysis of S-adenosylmethionine in animal tissues: the effect of exposure to nitrous oxide. Biomed. Chromatogr. 4:239-244.[Medline]
30. Durand, P., Fortin, L. J., Sussier-Cacan, S., Davignon, J. & Blache, D. (1996) Hyperhomocysteinemia induced by folic acid deficiency and methionine load-applications of a modified HPLC method. Clin. Chim. Acta 252:83-93.[Medline]
31.
Gupta, R. C. (1984) Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminoflourene to repetitive sequences of rat liver DNA in vivo.. Proc. Natl. Acad. Sci U.S.A. 81:6943-6947.
32.
Choi, S.-W., Stickel, F., Baik, H. W., Kim, Y.-I., Seitz, H. K. & Mason, J. B. (1999) Chronic alcohol consumption induces genomic but not p53-specific DNA hypomethylation in rat colon. J. Nutr. 129:1945-1950.
33. Cravo, M., Fidalgo, P., Pereira, A. D., Gouveia-Oliveira, A., Chaves, P., Selhub, J., Mason, J. B., Mira, F. C. & Leitao, C. N. (1994) DNA methylation as intermediate biomarker in colorectal cancer: modulation by folic acid supplementation. Eur. J. Cancer Prev. 3:473-479.[Medline]
34.
Kim, Y. I., Christmas, J. K., Fleet, J. C., Cravo, M. L., Salomon, R. N., Smith, D., Ordovas, J., Selhub, J. & Mason, J. B. (1995) Moderate folate deficiency does not cause global hypomethylation of hepatic and colonic DNA or c-myc specific hypomethylation of colonic DNA in rats. Am. J. Clin. Nutr. 61:1083-1090.
35.
Fiala, E. S., Staretz, M. E., Pandya, G. A., El-Bayoumy, K. & Hamilton, S. R. (1998) Inhibition of DNA cytosine methyltransferase by chemopreventive selenium compounds, determined by an improved assay for DNA cytosine methyltransferase and DNA cytosine methylation. Carcinogenesis 19:597-604.
36.
Cox, R. & Goorha, S. (1986) A study of the mechanism of selenite-induced hypomethylated DNA and differentiation of Friend erytrhroleukemic cells. Carcinogenesis 7:2015-2018.
37. Cox, R. (1985) Selenite: a good inhibitor of rat-liver DNA methylase. Biochem. Int. 10:63-69.[Medline]
38. Slack, A., Cervoni, N., Pinard, M. & Szyf, M. (1999) Feedback regulation of DNA methyltransferase gene expression by methylation. Eur. J. Biochem. 264:191-199.[Medline]
39.
Handel, M. L., Watts, C. K., deFazio, A., Day, R. O. & Sutherland, R. L. (1995) Inhibition of AP-1 binding and transcription by gold and selenium involving conserved cysteine residues in Jun and Fos. Proc. Natl. Acad. Sci. U.S.A. 92:4497-4501.
40. Spyrou, G., Bjornstedt, M., Kumar, S. & Holmgren, A. (1995) AP-1 DNA-binding activity is inhibited by selenite and selenodiglutathione. FEBS Lett 368:59-63.[Medline]
41. Uthus, E. O., Davis, C. D. & Yokoi, K. (2001) Selenium deprivation decreases the activity of liver betaine homocysteine methyltransferase in rats. FASEB 15:A969(abs.).
42. Le Leu, R. K, McIntosh, G. H. & Young, G. P. (1998) Ability of endogenous folate from soy protein isolate to maintain plasma homocysteine and hepatic DNA methylation during methyl group depletion in rats. J. Nutr. Sci. Vitaminol. 44:457-464.
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