Modulation by celecoxib and difluoromethylornithine of the methylation of DNA and the estrogen receptor-
gene in rat colon tumors
Michael A. Pereira1,3,
Lianhui Tao1,
Wei Wang1,
Yingzhe Li1,
Asad Umar2,
Vernon E. Steele2 and
Ronald A. Lubet2
1 Department of Pathology, Medical College of Ohio, Toledo, OH 43614, USA and 2 Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892, USA
3 To whom correspondence should be addressed Email: pereira-1{at}medctr.osu.edu
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Abstract
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The ability of celecoxib and
-difluoromethylornithine (DFMO) to modulate the DNA hypomethylation and the hypermethylation of the estrogen receptor (ER)-
gene in colon tumors was evaluated as potential biomarkers for chemoprevention. Colon tumors were induced in rats by azoxymethane. Celecoxib (500 mg/kg), DFMO (100, 1000 and 3000 mg/kg) or celecoxib + 1000 mg/kg DFMO were administered in the diet for 7 or 28 days prior to death at week 37. Relative to the normal colon mucosa, colon tumors contained global hypomethylated DNA but simultaneous hypermethylation of the promoter plus exon-1 region of the ER-
gene. Limited treatment with celecoxib (500 p.p.m. in diet) or DFMO (1000 or 3000 p.p.m. in diet) reversed the DNA hypomethylation. Administering 1000 and 3000 p.p.m. DFMO for 7-days decreased the number of methylated CpG sites in the ER-
gene from 5.00 ± 0.95 to 3.83 ± 0.75 and 1.75 ± 0.49 these levels were further reduced to 0.50 ± 0.26 following administration of 1000 mg/kg for 28 days. Celecoxib administered for 7 and 28 days reduced the number of methylated sites to 4.25 ± 0.48 and 1.5 ± 0.50. The combination containing celecoxib and DFMO reduced the number of methylated sites to 0.20 ± 0.20 at both 7 and 28 days. In parallel with the hypermethylation of the ER-
gene, the mRNA expression of the gene was decreased in colon tumors and was increased by celecoxib, DFMO or the combination. Celecoxib and DFMO reversed DNA hypomethylation and the hypermethylation of the ER-
gene in colon tumors supporting the hypothesis that modulation of methylation is a biomarker of chemoprevention.
Abbreviations: AOM, azoxymethane; DFMO,
-difluoromethylornithine; ER-
, estrogen receptor-
; 5-MeC, 5-methylcytosines
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Introduction
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Colon cancer is one of the major human cancers especially in the Western world including the US (1). About 56% of the US population has a lifetime risk of developing colon cancer. Because of its high incident and mortality rate, the identification of agents that prevent colon cancer would have a great public health benefit. Surrogate end-point biomarkers for chemoprevention are being developed for use in human clinical trials (24). Potential biomarkers include mechanism-based molecular alterations that can be modulated by short-term treatment with chemopreventive agents in parallel with their ability to prevent cancer. General DNA hypomethylation and the specific hypermethylation of tumor suppressor genes are examples of molecular alterations found in colon tumors (510). We hypothesized that chemopreventive agents might reverse the global DNA hypomethylation as well as the hypermethylation of tumor suppressor genes in colon tumors in parallel with their chemopreventive efficacy.
Approximately 4% of cytosine in mammalian DNA is methylated as 5-methylcytosines (5-MeC). General DNA hypomethylation is a common early event in most tumors, including both human colon cancer and chemically induced colon tumors in rodents (510). DNA hypomethylation has been proposed to have numerous consequences including decreased binding of methylated DNA-binding proteins, alteration in the binding and/or recruitment of transcription factors and enzymes involved in histone modifications, and increased chromosomal instability. The other alteration in methylation found in cancers, including colon cancer, is the hypermethylation of specific CpG-rich promoter regions in tumor suppressor genes (5,6). Hypermethylation of CpG islands in the regulatory region of genes, that usually includes exon-1 and its upstream region results in the silencing of the gene. The estrogen receptor-
(ER-
) gene was selected for the evaluation of the ability of chemopreventive agents to modulate the methylation of tumor suppressor genes in colon tumors. ER-
is a transcription factor that upon the binding of estrogen translocates to the nucleus where it activates various genes (1113). Hypermethylation of the ER-
gene has been reported in most human colon cancers (1417). In colon tumors, no other tumor suppressor gene approaches the very high incidence of hypermethylation that is found for the ER-
gene. The high incidence of colon tumors with hypermethylation of the ER-
gene should result in the ability to detect reversal of its hypermethylation by chemopreventive agents.
Azoxymethane (AOM)-induced colon tumors in rats is a standard bioassay for inducing colon tumors and has been used to identify and determine the efficacy of chemopreventive agents (18). Celecoxib and
-difluoromethylornithine (DFMO) have been shown to prevent AOM-induced colon tumors in this model (2026). In a previous study we had shown that short-term treatment with a variety of chemopreventive agents, including NSAIDS and DFMO, altered expression of various genes (25). The ability of short-term treatment with these two chemopreventive agents and a combination of both to modulate the generalized DNA hypomethylation and the specific hypermethylation of the ER-
gene was evaluated as potential surrogate end-point biomarkers for chemoprevention.
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Materials and methods
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Experimental protocol: administration to rats with AOM-induced colon tumors of chemopreventive agents for 7 and 28 days
Male F344 rats were obtained at 6 weeks of age from Charles River Laboratories (Frederick, MD). The rats were housed in the AAALAC-accredited laboratory animal facility at the Medical College of Ohio. Housing consisted of solid-bottomed polycarbonate cages with stainless steel wire-bar lids and Bed-o-Cob bedding (Andersons, Toledo, OH). The light cycle consisted of 12 h each of dark and light. Drinking water and AIN 76A diet (Dyets, Bethlehem, PA) were supplied to the animals ad libitum.
The rats at 78 weeks of age were randomly assigned to the various treatments groups of the study, so as to not bias the tumor response of the animals in the different groups. They were then administered 15 mg/kg AOM by i.p. injection, once a week for three consecutive weeks. Seven and 28 days prior to death at 37 weeks after the first dose of AOM, the rats were administered in their diet either celecoxib at 500 mg/kg, DFMO at 100, 1000 and 3000 mg/kg or a combination containing 500 mg/kg celecoxib and 1000 mg/kg DFMO according to their prior designated treatment group. At necropsy, colon tumors were harvested, rapidly frozen in liquid nitrogen and stored at 70°C. The majority of colon tumors at 37 weeks are adenomas with the remainder being adenocarcinomas.
Eight age-control rats were used to isolate colonic mucosa. These rats were from the same shipment, did not receive any treatment and were killed at the same time as the other rats of this study. At death their colons were harvested, frozen flat on dry ice and then scraped to obtain the mucosa. The isolated mucosa was refrozen using liquid nitrogen and stored at 70°C.
DNA methylation: dot blot analysis
DNA was isolated by digestion with RNase A and proteinase K followed by organic extraction (27). Purified DNA (2 µg) was denatured with 0.1 N NaOH at 100°C for 5 min, neutralized with 2 M ammonium acetate and applied onto HybondTM nitrocellulose membranes using a Bio-Dot Microfiltration Apparatus (Bio-Rad Laboratories, Hercules, CA). The membranes were heated under vacuum at 80°C for 2 h followed by treatment with 5% milk dissolved in Tris-buffered saline + Tween-20 (TBST) blocking solution (pH 7.6) for 2 h. They were then incubated with a 1:1000 dilution of mouse monoclonal antibody against 5-MeC (Eurogentec Company, Belgium) for 2 h, washed with TBST (pH 7.6) and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated anti-mouse-IgG antibody for 1 h. The membranes were washed again with TBST (pH 7.6), treated with enhanced-chemiluminescence western blotting detection reagents and exposed to Kodak autoradiograph films. Optical density of the dots was measured with the Scion Image Analysis System (Scion, Frederick, MD). Equal loading of the DNA onto the membrane was indicated by equal intensity of methylene blue stained dots. The optimum dilution of the 5-MeC antibody was determined previously to be 1:1000 and the specificity of the antibody was demonstrated by the ability of 5-MeC but not cytosine to block the antibody (28).
Methylation of the ER-
gene: bisulfite treatment followed by sequencing
DNA was isolated as described above for the dot blot analysis. DNA (2 µg) was denatured with 0.3 N NaOH for 20 min at 37°C. Freshly prepared 2.2 mol/l sodium metabisulfite/0.5 mmol/l hydroquninone mixture (pH 5) was added and the solution incubated under mineral oil at 50°C for 16 h. The bisulfite-modified DNA was recovered using a mini-Sephadex G-50 gel column. The DNA was again treated with 0.3 N NaOH for 20 min at 37°C and neutralized with 10 M ammonium acetate. Glycogen was added as a carrier and the DNA precipitated with ethanol. The PCR primers for exon-1 and its 5'-flanking region of the ER-
gene were upstream: 5'-TTTTAGGAATGTTGATTTTAGTGGT-3' (nt 19992023) and downstream: 5'-AACACAACCTCCTTC TCCAACTA-3' (nt 24122434) (GenBank Database Accession Number X98236). The PCR reaction consisted of 2 min at 94°C, 5 min at 59°C, 10 min at 72°C, and 44 cycles consisting of 1 min at 94°C, 1 min at 59°C and 1.5 min at 72°C. The PCR product was electrophoresed in 1% agarose gel containing 0.1 µg/ml ethidium bromide. The band at
436 bp was cut out, extracted using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), and ligated into Topo TA cloning vector using standard protocols (Invitrogen, Carlsbad, CA). Plasmid DNA containing the PCR product was sequenced using forward primer: 5'-TTGGTACCGAGCTCGGAT-3' and reverse primer: 5'-CGTAGATCT CCCGGGTTA-3'.
mRNA expression of ER-
RNA was isolated using the TriZol extraction protocol. RTPCR was performed using 1 µg of total RNA that was added to 20 µl of reverse transcriptase buffer (50 mM potassium chloride, 5 mM MgCl2 and 10 mM TrisHCl, pH 9.0) containing 0.5 µg oligo(dT)15 primer, 15 U avian myeloblastosis virus reverse transcriptase, 1 µg/µl RNasin inhibitor, and 1.0 mM of each of the four deoxynucleoside triphosphates. The reaction mixture was incubated for 40 min at 42°C, 5 min at 99°C and 5 min at 4°C. The subsequent PCR was carried out in 50 µl containing 200 pmol of the forward 5'-TGACCCTTCACACCAAAGCCT-3' (nt 45154535) and reverse 5'-ATCAGCGGACTGGGCGACA-3' (nt 48304848) primers, specific for exon-1 of the ER-
gene (GenBank Database Accession Number X98236). The PCR reaction consisted of 30 cycles of 45 s at 94°C, 45 s at 60°C and 60 s at 72°C. This was followed by 10 min at 72°C. As a control, GAPDH (GenBank Database Accession Number AF106860) was co-amplified with the ER-
gene. The upstream primer for GAPDH was 5'-ATGGTGAAGGTCGGTGTGAACG-3' (nt 850871) and the downstream primer was 5'-GTTGTCATGGATGACCTTGGCC-3' (nt 13231344). The PCR products were electrophoresed in 1% agarose gel containing 0.1 µg/ml ethidium bromide. After electrophoresis, the gels were photographed under UV-irradiation and the optical density of the mRNA bands measured with the Scion Image Analysis System. The optical density of the mRNA for the ER-
gene was standardized using the density of the GAPDH product.
Statistical analysis
Results were analyzed for statistical significance by either the t-test or by a one-way analysis of variance (ANOVA) followed by the Bonferroni t-test. A P-value <0.05 was considered statistically significant. Data are expressed as means ± SE.
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Results
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DNA hypomethylation in colon tumors
The DNA in colon tumors relative to colon mucosa was hypomethylated; the level of 5-MeC was decreased by 6075% (Figure 1). Administering celecoxib and DFMO for only 7 days prior to death reversed the DNA hypomethylation in tumors. DFMO caused a dose-dependent increase in DNA methylation with 1000 and 3000 mg/kg being effective, while 100 mg/kg was ineffective. Both celecoxib and 1000 mg/kg DFMO were more effective in reversing DNA hypomethylation after 28 days of treatment than after 7 days and after 28 days of treatment the level of DNA methylation in the tumors was no longer different from the level in normal mucosa.

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Fig. 1. Effect of DFMO and celecoxib on DNA methylation in rat colon tumors. (A) Representative dot blot analysis using the antibody to 5-MeC in DNA. The numbers above the dots are individual animal numbers. The membranes were stained for the 5-MeC antibody and then with methylene blue to indicate equal loading of DNA. (B) DFMO and (C) celecoxib. Optical density of the dots was measured with the Scion Image Analysis System and is presented as mean ± SE for four tumors from different animals. Bars with different letters were statistically different, P-value <0.05.
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Methylation of ER-
gene in colon tumors
After bisulfite treatment, the methylation status of the ER-
gene was determined for 19 CpG sites in the beginning of exon-1 and its 5'-upstream region. Only one of the 19 sites was methylated in DNA in a single sample isolated from the mucosa of eight age-control rats. All colon tumors had at least two methylated sites and eight of eleven had at least five methylated CpG sites. There was an average of 5.00 ± 0.95 methylated sites, which was strikingly greater than the mean of 0.13 ± 0.13 methylated sites in normal colon mucosa (P-value <0.001). The number of methylated CpG sites was reproducibly higher in exon-1 than in the proximal promoter region.
DFMO treatment decreased the number of methylated sites in the evaluated region of the ER-
gene (Table I). The mean number of methylated sites was 5.00 ± 0.95, 7.33 ± 0.33, 3.83 ± 0.75 and 1.75 ± 0.49 for rats administered 0, 100, 1000 or 3000 mg/kg DFMO for 7-days, with the decrease resulting from 3000 mg/kg treatment being statistically significant. Administering 1000 mg/kg DFMO for 28 days decreased the number of methylated sites to 0.50 ± 0.26 that was significantly different from colon tumors of rats not administered DFMO and of rats administered 1000 mg/kg for only 7 days (P-value <0.001). Thus, DFMO decreased the number of methylated sites in the ER-
gene, causing a greater reduction after 28 days than after 7 days.
Treatment with a suboptimal chemoprevention dose of celecoxib (500 p.p.m.) significantly reduced the number of methylated sites in the ER-
gene from 5.00 ± 0.95 to 1.5 ± 0.50 after 28 days of treatment, while after only 7 days of treatment the number of methylated sites was not significantly reduced being 4.25 ± 0.48 (Tables II and III). Combined treatment with both celecoxib (500 mg/kg) and DFMO (1000 mg/kg) significantly reduced the number of methylated sites to 0.20 ± 0.20 after both 7 and 28 days of treatment (P-value <0.001). Thus, 7 days of treatment with this combination was significantly more effective in reducing the number of methylated sites than either celecoxib or DFMO administered alone.
mRNA expression of the ER-
gene
The mRNA expression of the ER-
gene was determined by RTPCR with the GAPDH gene co-amplified as a control (Figure 2). The mRNA expression of the ER-
gene was decreased in colon tumors by
50% relative to the normal mucosa. Treatment with 500 mg/kg celecoxib, 1000 mg/kg DFMO or their combination for 7 or 28 days increased the expression of the gene to at least the level found in the normal mucosa. In fact, the expression of the gene after treatment with DFMO with/without co-administered celecoxib was greater in colon tumors than in normal mucosa. The results support an association between methylation in the promoter and exon-1 region of the ER-
gene and the mRNA expression of the gene, so that hypermethylation of the gene results in deceased mRNA expression and reversal of the hypermethylation results in increased mRNA expression.
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Discussion
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The modulation of surrogate end-point biomarkers by short-term treatment with chemopreventive agents has the potential of being used in animal studies and clinical trials to screen for active agents. This is especially true for surrogate end-point biomarkers that are related to the molecular mechanisms of colon carcinogenesis and are modulated in concordance with the preventive efficacy of the agents in decreasing cancer incidence. DNA hypomethylation is an early event in the neoplastic progression of most human and animal cancers including colon cancer (510). In a previous publication we reported that DNA in AOM-induced rat colon tumors was hypomethylated relative to colonic mucosa (28). We have also reported that DNA hypomethylation in colon tumors is reversed by various chemopreventive agents. Only 7 days of treatment with agents that prevent colon cancer in rats, including calcium chloride, DFMO, piroxicam and sulindac was sufficient to reverse the DNA hypomethylation in rat colon tumors. In contrast, agents that do not prevent colon cancer in rats did not reverse the hypomethylation (28). We now report that celecoxib, another drug that prevents colon cancer in rats (2023), also reverses DNA hypomethylation within 7 days. The ability to modulate the methylation status of DNA can also be used to distinguish agents that are effective in preventing colon cancer from those that are ineffective and further, that reversal of DNA hypomethylation is a potential biomarker for identifying agents that prevent colon cancer. Although these agents are strong preventive agents when administered long term they do not appear to be highly effective therapeutic agents in the lesions examined after 7 or 28 days of treatment. Thus, these treated lesions appear normal both with regard to size and histopathology.
One of the advantages of DNA hypomethylation as a surrogate end-point biomarker is that chemopreventive agents of different mechanisms including calcium, DFMO, the non-specific cyclooxygenase-1 and -2 inhibitors piroxicam and sulindac as well as the specific COX-2 inhibitor, celecoxib can reverse it in colon tumors. A second advantage is that DNA hypomethylation is a molecular alteration found in many types of tumors and may be a biomarker applicable to many other cancers besides colon cancer. Furthermore, the procedure for analyzing DNA methylation is not organ specific and therefore is applicable to cancer of different organs.
The reversal of DNA hypomethylation by DFMO was dependent on the dose and duration of treatment. The doseresponse of DFMO in reversing DNA hypomethylation was similar to its doseresponse in preventing colon cancer. DNA hypomethylation was reversed by dose levels of 1000 and 3000 mg/kg of DFMO that have been shown previously to prevent colon cancer (2426), but not by 100 mg/kg DFMO. As part of a doseresponse study, 400 mg/kg DFMO in the diet, the lowest dose evaluated was reported to cause a minimal, but statistically significant decrease in tumor multiplicity, so one would expect that 100 mg/kg DFMO would not be sufficient to prevent colon tumors (26). Furthermore, it would appear that >7 days of treatment, possibly 28 days are required to obtain optimum reversal of DNA hypomethylation by DFMO and celecoxib.
Another alteration in methylation found in tumors is the hypermethylation of CpG-rich promoter regions in tumor suppressor genes (5,6). The specific genes, which are hypermethylated vary with tumor type. This alteration in methylation is also being evaluated for use as a surrogate end-point biomarker for chemoprevention. One potential appeal of examining hypermethylation and its reversal by agents is that one may be able to produce a sensitive assay for identifying hypermethylated sites in a gene. Hypermethylation of the ER-
gene has been reported in most if not all human colon cancers (1417). Expression of the two estrogen receptors, ER-
and ER-ß, has been reported in colon mucosa and may mediate estrogen-associated reduction of colon cancer susceptibility (2931). Expression of ER-
in colon mucosa and cancers is low relative to ER-ß. Issa et al. (13) have reported that hypermethylation of the ER-
promoter increased as a function of age and suggested that ER-
was a tumor suppressor gene in the colon. Age-related hypermethylation of other tumor suppressor genes has subsequently been reported (1417). Reversal or prevention of the hypermethylation of ER-
and other tumor suppressor genes in the colon resulting in their increased expression could contribute to a delay and to a decrease in the risk of cancer.
We have found that the ER-
gene was hypermethylated in AOM-induced colon cancer, demonstrating it to be a molecular alteration that this rat model has in common with human colon cancer. Two effective chemopreventive agents for colon cancer, celecoxib and DFMO caused significant reversal of the hypermethylation of the ER-
gene, particularly in exon-1. The major pharmacological mechanism of celecoxib and DFMO is quite different with celecoxib being a selective COX-2 inhibitor and DFMO an ornithine decarboxylase inhibitor, yet both were highly effective in reversing the hypermethylation of the ER-
gene. Hence, similar to the reversal of DNA hypomethylation, reversal of the hypermethylation of this gene in colon tumors would appear to be a biomarker for chemopreventive agents of different mechanisms.
The ability to reverse the hypermethylation of the ER-
gene was also dose-dependent. Similar to the reversal of DNA hypomethylation, dose levels (1000 and 3000 mg/kg) of DFMO that prevent colon cancer were effective in reversing the hypermethylation, while 100 mg/kg DFMO, a dose that appears not to prevent colon cancer in rats was not effective. The dose of celecoxib (500 mg/kg diet) that reversed the hypermethylation of the ER-
gene has been reported by Reddy et al. (23) to prevent AOM-induced colon tumors in rats. In fact, higher concentrations of celecoxib in the diet, i.e. 1000 and 1500 mg/kg were more effective in preventing colon cancer. Thus, we were able to demonstrate that celecoxib reversed the hypermethylation of the ER-
gene at a less than maximum effective chemopreventive dose. It seems plausible to expect that short-term treatment with a higher dose of celecoxib that was more effective as a chemopreventive agent might affect hypermethylation to a greater degree. A combination containing 500 mg/kg celecoxib and 1000 mg/kg DFMO was more efficacious than the individual drugs in reversing the hypermethylation of the ER-
gene. The greater efficacy of the combination of celecoxib and DFMO in reversing hypermethylation suggests that it might have a greater efficacy in preventing colon cancer than the individual agents. Another NSAID, piroxicam, in combination with DFMO has been shown to be more efficacious than the individual drugs in preventing both AOM-induced colon cancer and spontaneous intestinal cancers in Min mice (26,32). The combination of celecoxib and DFMO has recently been shown to be more effective than either agent alone in preventing UV-induced skin cancers in SKH-1 mice (33). This would further support the suggestion that the combination of celecoxib and DFMO might have a greater efficacy than either drug in preventing colon cancer. Furthermore, the dose dependency of DFMO and the greater efficacy of the combination to reverse hypermethylation of the ER-
gene indicate that reversal of hypermethylation correlates with prevention of colon cancer in rats.
Our results are not meant to imply that reversal of the alterations in methylation in colon tumors is a direct manifestation of the chemopreventive agents. The mechanism is not known by which chemopreventive agents simultaneously can reverse global DNA hypomethylation and the specific hypermethylation of the ER-
gene. Since chemopreventive agents of various pharmacological mechanisms reversed these alterations in methylation, it is probable that the molecular mechanism of reversal is related to general molecular alterations caused by these agents and not to a specific pathway. Thus, we have demonstrated previously that treatment with chemopreventive agents modulated the expression of a variety of genes (e.g. c-myc, p16, p21 and p27) in colon tumors towards expression levels found in normal mucosa following limited exposure (25). One mechanism by which chemopreventive agents could reverse DNA hypomethylation in tumors is to increase the activity of DNA methyltransferase (Dnmt) and/or increase the level of the methyl donor, S-adenosyl methionine (SAM). However, neither an increased level of Dnmt nor of SAM would explain the concurrent reversal of the hypermethylation of ER-
gene simultaneously with the reversal of generalized DNA hypomethylation. Furthermore, it would appear that reversal of hypermethylation by celecoxib and DFMO is not the result of them directly blocking methylation. Their ability to simultaneously reverse global DNA hypomethylation implies that blocking DNA methylation is not their primary mode of action.
A mechanism for the reversal of the hypermethylation of the ER-
gene by chemopreventive agents could involve histone acetylation, transcription factors and other factors that bind to DNA. It is possible that chemopreventive agents could reverse the hypermethylation of the ER-
gene by decreasing histone acetylation. Close to the region of the ER-
gene that we have shown to be hypermethylated, there are two E-boxes (CACGTG) that bind the transcription factor, Myc. Myc has been shown to recruit histone acetylase to increase histone acetylation (34). This Myc directed increase in histone acetylation could open up the chromatin in the vicinity of the two E-boxes and allow nearby CpG sites that were unmethylated previously to become methylated. We have shown that the c-myc gene in colon tumors is down-regulated by DFMO, NSAIDS and other chemopreventive agents (25). The down-regulation of the c-myc gene should decrease its binding to the two E-boxes resulting in decreased recruitment of histone acetylase, decreased histone acetylation and condensing of the surrounding chromatin to decrease the methylation of the ER-
gene. However, such a specific mechanism would not simultaneously account for the reversal of global DNA hypomethylation. Although the present study does not present a clear mechanism by which chemopreventive agents modulate the methylation of DNA and the ER-
gene in colon tumors, the study does identify two surrogate end-point biomarkers for chemoprevention in the colon, i.e. reversal of global DNA hypomethylation and the hypermethylation of tumor suppressor genes.
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Acknowledgments
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Supported in part by National Cancer Institute contracts N01-CN-05123 and N01-CN-15121.
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Received February 13, 2004;
revised May 18, 2004;
accepted June 6, 2004.