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1 Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,
2 Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey
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ABSTRACT |
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Introduction |
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Hypermethylation of DNA is a key epigenetic mechanism for the silencing of many genes, including those for cell cycle regulation, receptors, DNA repair, and apoptosis (5, 6, 7, 8) . In this aberrant methylation, the cytosine of the CpG island in or near the promoter region of the newly synthesized DNA strand is methylated by DNMT. The methylated CpG island has higher binding affinity to methyl-CpG binding domain proteins that recruit transcriptional corepressors, such as histone deacetylases and Sin 3A, resulting in chromosome condensation and transcription repression (9 , 10) . The inhibition of DNMT, especially DNMT1, would block the hypermethylation of the newly synthesized DNA strand, resulting in the reversal of the hypermethylation and the re-expression of the silenced genes (11, 12, 13) . Indeed, this point has been demonstrated by studies with DNMT inhibitors, DAC (also known as 5-aza-2'-deoxycytidine) and zebularine. These compounds have been shown to inhibit cancer cell growth, induce cancer cell apoptosis, and reduce tumor volume in mice (14, 15, 16, 17) . There is high potential for developing this group of inhibitors for cancer therapy. However, side effects and toxicity are serious concerns. There is a great need for the development of effective and nontoxic inhibitors of DNMT. We hypothesize that EGCG can be used for this purpose.
In the present study, we tested this hypothesis by examining the inhibition of DNMT by EGCG and the effects of EGCG treatment on four methylation-silenced genes in human esophageal cancer cell line KYSE 510: the tumor suppressor p16INK4a, retinoic acid receptor ß (RARß), O6-methylguanine methyltransferase (MGMT), and the DNA mismatch repair gene human mutL homologue 1 (hMLH1). In our previous work, these genes have been shown to be frequently silenced by DNA hypermethylation in human esophageal cancer, and some of these genes can be partially reactivated by treatment with the DNMT inhibitor, DAC (18) . The results on the inhibition of DNMT and reactivation of methylation-silenced genes by EGCG are reported herein.
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Materials and Methods |
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DNA Methyltransferase Assay.
Cultured KYSE 510 cells were harvested, and nuclear extracts were prepared with the nuclear extraction reagent (Pierce, Rockford, IL). The DNMT assay was performed according to published methods (19
, 20)
. In brief, the nuclear extracts (4 µg of protein) were incubated for 1.5 or 2 h at 37°C with 0.66 µM of poly(dI-dC)·poly(dI-dC) and 10 µM of S-adenosyl-L-[methyl-3H]methionine (2 µCi; Amersham, Piscataway, NJ) in a total volume of 40 µl of a pH 7.4 buffer, containing 20 mM Tris-HCl, 25% glycerol (v/v), 10 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 20 mM 2-mercaptoethanol. All of the incubations contained 0.02% DMSO, which was used to dissolve EGCG and other inhibitors. Reactions were initiated by the addition of nuclear extracts and stopped by mixing with 300 µl of a solution containing 1% SDS, 3% 4-aminosalicylate, 5% butanol, 2.0 mM EDTA, 125 mM NaCl, 0.25 mg/ml carrier salmon testes DNA (Life Technologies, Inc., Gaithersburg, MD), and 1 mg/ml protease K (Sigma, St. Louis, MO). To remove proteins, the resulting mixture was vortexed with 300 µl of a solution containing 8% phenol, 12% m-cresol, and 0.1% 8-hydroxyquinoline (Sigma), and centrifuged. The methylated DNA template was recovered from the aqueous phase by ethanol precipitation and then washed three times with 70% ethanol. The radioactivity in the pellets was counted in a scintillation counter. All of the assays were performed in duplicate. Background levels were determined in incubations without the template DNA.
In Silico Molecular Modeling Studies of DNMT1.
The protein sequence of DNMT1 (accession no. NP_001370) was retrieved from the National Center for Biotechnology Information Reference Sequence (RefSeq) Collection. A structural model of the catalytic domain of DNMT1 was constructed using the Insight II Homology Module (Accelrys, Inc., San Diego, CA) from the published crystal structure of HhaI Mtase (RCSB Protein Data Bank;4
PDB ID = 5MHT) as the modeling template. The quality of the model was confirmed by the WHATIF-Check program.5
Initial structures of EGCG and the five analogues were built using the Insight II Builder Module and subsequently energy minimized to yield a stable conformation. Each ligand was docked into the putative DNMT1 binding pocket using GOLD (Genetic Optimization for Ligand Docking; Ref. 21
), which accommodates ligand flexibility and partial enzyme flexibility to satisfy ligand-enzyme binding requirements. After these docking studies to elucidate stable ligand-binding modes, values of the ligand-enzyme binding energy (
Ebinding) were calculated for each of the six ligands using molecular mechanics procedures. Residues within 5.0 Å of the cytosine nucleotide were defined as the "active site" pocket and were permitted full relaxation, while holding all of the other residues fixed. We define Ebinding as the difference between the potential energy of the ligand-enzyme (Ecomplex) and the sum of potential energies of the ligand (Eligand) and enzyme (Eenzyme).
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Bisulfite Modification and Methylation-Specific PCR.
DNA was extracted from the cells using the DNeasy tissue kit (Qiagen, Valencia, CA), and was denatured with 0.2 M NaOH at 37°C for 30 min. The denatured DNA was treated with 3.3 M sodium bisulfite and 0.66 mM hydroquinone according to Herman et al. (22)
, with slight modifications (18)
. The modified DNA was purified using the Qiaquick gel extraction kit (Qiagen) and eluted with water. NaOH was added to the modified DNA for a final concentration of 0.3 M and maintained for 5 min at room temperature to desulfonate the modified DNA. The resulting DNA was repurified with Qiaquick gel and used immediately or stored at -20°C.
Methylation-specific PCR was carried out using a nested two-stage PCR approach (23)
. Stage I PCR was performed on bisulfite-modified DNA to amplify a 208-, 289-, 425-, or 185-bp fragment of the CpG-rich promoter regions of the p16INK4a, MGMT, RARß, or hMLH1 genes, respectively. The primers recognize the bisulfite-modified template but do not discriminate between methylated and unmethylated alleles. The stage I PCR products were diluted and subjected to a stage II PCR with primer sets specific to methylated or unmethylated template. The primers were designed according to the literature (23, 24, 25, 26, 27)
. The chromosomal locations of all of the genes tested, primer sequences for both the methylated and unmethylated templates, annealing temperatures used, and expected PCR product sizes are summarized in Table 1
. Placental DNA and placental DNA treated with SssI methyltransferase (New England Biolabs, Beverly, MA) were used as positive controls for unmethylated alleles and methylated alleles, respectively. Amplification was carried out using a 9700 Perkin-Elmer thermal cycler (Applied Biosystems, Foster City, CA).
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. The PCR reactions were run in an approximate linear range (for example, 25–28 cycles for p16INK4a, 30–35 cycles for RARß, 20–25 cycles for hMLH1, and 30–35 cycles for MGMT). The amplified products were subjected to electrophoresis on a 2% agarose gel. The gel was then stained with ethidium bromide and photographed. Negative controls for PCR were run under the same conditions without RNA or reverse transcriptase. Expression of the glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH) was used to examine the integrity of RNA in each sample and to standardize the amount of cDNA added to each of the PCR reactions.
Western Blot.
The protein samples in SDS-containing lysis buffer were loaded on 4–15% acrylamide gel (40 µg/lane). After electrophoresis, the proteins were blotted onto a nitrocellulose membrane. After incubation in blocking buffer (5% nonfat milk), the membranes were incubated with the primary antibodies (rabbit antihuman RARß and hMLH1 polyclonal antibodies; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. After washing with Tris-buffered saline containing 0.1% Tween 20, the membrane was then incubated with sheep antirabbit horseradish peroxidase-labeled secondary antibody and visualized using the electrochemiluminescense detection kit (Amersham).
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Results |
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20 µM (Fig. 1A)
. The structural analogues of EGCG: ECG, EGC, EC, and methylated EGCG all had inhibitory activities, with potency in the rank order of EGCG > ECG, MeEGCG > EGC, and DiMeEGCG > EC. Kinetic studies indicated that EGCG increases the Km of DNMT without affecting the Vmax, suggesting a competitive inhibition with a Ki of 6.89 µM (Fig. 1B)
. The same conclusion was reached when this experiment was repeated.
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. Docking of EGCG into the putative cytosine pocket indicated that the gallate moiety of this compound formed potential hydrogen bonds with two catalytically important residues, Glu1265 and Pro1223, which were the same residues that appear to stabilize the flipped cytosine through hydrogen bonding. Interestingly, the D ring was oriented at approximately the same position as the pyrimidyl ring of cytosine in the structural model of DNMT1. Furthermore, Ser1229 and Cys1225 are positioned to form hydrogen bonds with the hydroxy groups in the A and B rings of EGCG, respectively, thereby contributing to high-affinity DNMT1 binding. The interactions are depicted in Fig. 2B
. These findings suggest that EGCG exerts its inhibitory effect on DNMT1 function by blocking entry of the key nucleotide cytosine into its active site and, thus, prevents methylation. A consensus-binding mode was obtained from 25 independent docking procedures for EGCG and each of the five analogues bound to DNMT1. Accordingly, calculated values of Ebinding (shown in parentheses, in units of kcal/mol) for each ligand in its consensus-binding mode yielded the following order of binding affinity to DNMT1: EGCG (-49.75) > ECG (-46.53)> Me-EGCG (-44.91) > diMe-EGCG (-41.64) > EGC (-40.79) > EC (-38.32). These predicted results are generally consistent with the experimental data.
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. Corresponding to the appearance of the unmethylation-specific band was the re-expression of mRNA of all four of the genes. The unmethylation-specific bands and mRNA expression were both observed with 20 and 50 µM of EGCG. For hMLH1, the unmethylation-specific band was observed even with 10 µM of EGCG in comparison to the faint bands observed with 0 and 5 µM EGCG (Fig. 3A)
. The reversal of hypermethylation and re-expression of these four genes by EGCG was similar to that produced by the classical DNMT inhibitor, DAC. After treating the cells with 20 µM of EGCG for 0, 12, 24, 48, 72, and 144 h, strong unmethylation-specific bands for all four of the genes began to appear at 48 h (Fig. 3A)
. The mRNA expression of these four genes was also observed at 48 h and, depending on the genes, higher levels were observed at either 72 or 144 h. Again, a faint hMLH1 mRNA band was observed at 0, 12, and 24 h. Similar results were obtained in two repeated experiments.
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. In another experiment, demethylation and reactivation of RARß was observed with DiMeEGCG at 50 µM, but not at 20 µM (data not shown).
Western blot analysis indicated that the RARß protein expression was increased in the cells treated with 20 and 50 µM of EGCG for 6 days (Fig. 4)
. A time-dependent increase of hMLH1 protein expression level was observed in the cells treated with 20 µM of EGCG for 3 and 6 days. Because of the lack of suitable antibodies, we were unable to accurately analyze the re-expression of MGMT and p16INK4a proteins.
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. The appearance of unmethylation-specific bands and reactivation of the mRNA of p16INK4a in HT-29 cells, and RARß in KYSE 150 and PC3 cells, were observed after treating the cells with 20 µM EGCG for 6 days. In the KYSE 150 cells, a faint RARß mRNA band was also observed in cells without EGCG treatment, but the band was much more intense after EGCG treatment. These results were reproduced in three experiments with the three cell lines. The results, although still preliminary in a quantitative sense, demonstrate that the reactivation of methylation-silenced genes does occur in different cell lines, suggesting it is a general phenomenon.
Inhibition of Cell Growth and Other Effects.
After treatment with EGCG for 6 days, significant inhibition of cell growth was not observed at 5 or 10 µM, but 36 and 54% inhibition were observed at 20 and 50 µM of EGCG, respectively (Fig. 5A)
. Many damaged cells and some polynuclear cells were observed with 50 µM of EGCG, and to a lesser extent with 20 µM of EGCG, in this 6-day experiment (Fig. 5C)
. In experiments in which cells were treated for 2 days with EGCG, growth inhibition and signs of toxicity were not apparent at 5, 10, or 20 µM; but at 50 µM, some damaged cells with vesicles were observed (Fig. 5C)
. After treatment of the cells with 50 µM of EGCG for 2 days, the colony formation (measured after an additional 7 days of culturing) was inhibited by 55%, but significant inhibition was not observed with EGCG at 20 µM or lower concentrations (Fig. 5B)
. Preliminary experiments with RT-PCR also indicated that EGCG treatment did not affect the mRNA expression level of DNMT1, DNMT3a, DNMT3b, and MBD2 (methyl-CpG binding domain protein 2), which has been reported to have DNA demethylase activity (31)
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Discussion |
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, EGCG is effectively tethered within the DNMT1 binding site by at least four hydrogen bonds involving Ser1229 (2.5Å), Glu1265 (3.1Å), Pro1223 (3.0 Å), and Cys1225 (3.1Å). An additional hydrogen bond is possible with the protonated side chain of Arg1309, although the N... O distance (3.8Å) is at the outer limit of the typical hydrogen bond and, thus, would imply a weak interaction. This hydrogen-bonding scheme is consistent with the observed reduction in DNMT1 inhibitory activity for the five EGCG structural analogues. The absence of the B-ring 3'-hydroxy group in ECG abolishes the hydrogen bond with Cys1225. Likewise, the absence of the 4''-hydroxy group in MeEGCG eliminates the hydrogen bond with Pro1223. Addition of another methyl group at the B ring as in DiMeEGCG additionally interferes with the binding. The importance of galloyl moiety in EGCG-like compounds is evident from interactions with the two conserved residues, Glu1265 and Pro1223, which are associated with the catalytic activity of DNMT1. This observation concurs with the experimental results for EGC and EC, two poor DNMT1 inhibitors in which the galloyl moiety is conspicuously absent. The activities of these compounds to reactivate RARß mRNA expression are also correlated with their ability to inhibit DNMT activity. With EGCG, demethylation and reactivation of all four of the genes are observable after 48 h of treatment, and 20 µM is the effective concentration that activates all four of the genes. All of the results are consistent with our hypothesis that reactivation of methylation-silenced genes is due to the inhibition of DNMT by EGCG. More work is needed to determine the quantitative reactivation of the different genes as well as the extents of the demethylation in different cell lines under different treatment conditions. It would be interesting to determine whether the different hypermethylated genes respond similarly or differently to the EGCG treatment. The reactivated p16INK4a is expected to regain its function in cell cycle regulation, and re-expressing RARß is expected to regain responsiveness to retinoic acids. It is not clear whether the presently observed growth -inhibitory effect after 2 or 6 days of treatment with EGCG are related to the reactivation of these genes. The functional importance of the reactivation of those genes remains to be determined.
Hypermethylation of DNA is a key epigenetic mechanism for the silencing of many genes including those for tumor suppressors, DNA repair enzymes, and receptors (5, 6, 7, 8) . In theory, this epigenetic process is reversible if the newly synthesized DNA strands are not methylated. Therefore, DNMT inhibitors such as DAC and zebularine have been actively explored as cancer therapeutic agents, especially when they are used in combination with a histone deacetylase inhibitor, such as trichostatin A (15 , 26 , 32, 33, 34, 35, 36) . It has also been suggested that methylation is not the initial event in triggering gene silencing in cancer; rather, the methylation of the promoter CpG islands is a consequence of prior gene inactivation, and it is a mechanism for locking the chromatin in a repressed state (5 , 13 , 37) . Many tumor suppressor and receptor genes have been reported to be hypermethylated and transcriptionally silenced during the development of different types of cancers. It is likely that inhibition of DNMT and histone deacetylase can also prevent the hypermethylation and silencing of these key genes and, therefore, this inhibition would contribute to the prevention of carcinogenesis. The prevention of intestinal tumorigenesis by Dnmt1 deficiency and by DAC has been demonstrated in the Min mice, which carry a mutated Apc gene (38 , 39) .
Oral administration of green tea has been shown to inhibit tumorigenesis in different organs, and multiple mechanisms may be involved (1) . It remains to be determined whether and to what extent this cancer-preventive activity is due to the inhibition of DNMT by EGCG. The presently observed effective dose of EGCG, Ki of 6.89 µM, or IC50 of 20 µM, is achievable in the oral cavity (in the saliva) after drinking green tea, and perhaps in the stomach, esophagus, and intestines where there is direct contact between EGCG and the epithelial cells. This effective concentration, however, is higher than those in the internal organs, which depend on the systemic bioavailability of EGCG (1) . Therefore, the extent of DNMT inhibition in vivo would depend on the bioavailability of EGCG in a particular organ site. Although inhibition of DNMT is expected to prevent hypermethylation, severe inhibition of DNMT activity, as suggested by recent genetic studies, may cause DNA hypomethylation, genomic instability, and early development of cancers such as T-cell lymphomas and sarcomas (40 , 41) . There is no evidence for such adverse effects due to regular consumption of tea; however, it could be a concern if large doses of EGCG are given to humans.
On the basis of this analysis, for the practical application of tea in the prevention of cancer, it may be desirable to use doses to produce levels of EGCG in the tissue that would cause a moderate inhibition of DNMT. The possible synergy generated between EGCG with histone deacetylase inhibitors or other agents will hold great promise. For example, butyrate, which is a histone deacetylase inhibitor produced in the colon (42) , may work synergistically with EGCG in the prevention of colon cancer. The application of this concept in animal models or humans remains to be demonstrated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
M. Z. F. and Y. W. contributed equally to this work.
Requests for reprints: Chung S. Yang, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020. Phone: (732) 445-5360; Fax: (732) 445-0687; E-mail: csyang{at}rci.rutgers.edu
3 The abbreviations used are: EGCG, (-)-epigallocatechin-3-gallate; MeEGCG, 4''-O-methyl-EGCG; DiMeEGCG, 4', 4''-O-dimethyl-EGCG; DNMT, 5-cytosine DNA methyltransferase; DAC, 2'-deoxy-5-azacytidine; RARß, retinoic acid receptor ß; MGMT, O6-methylguanine methyltransferase; hMLH1, human mutL homolog 1; poly(dI-dC)·poly(dI-dC), polydeoxyinosine-deoxycytosine; ECG, (-)-epicatechin gallate; EGC, (-)-epigallocatechin; EC, (-)-epicatechin; RT-PCR, reverse transcription-PCR. 
4 Internet address: http://www.rcsb.org/pdb.
5 Internet address: http://www.cmbi.kun.nl/gv/servers/WIWWWI.
Received 5/29/03. Revised 9/ 8/03. Accepted 10/ 6/03.
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J. C. Chuang, C. B. Yoo, J. M. Kwan, T. W.H. Li, G. Liang, A. S. Yang, and P. A. Jones Comparison of biological effects of non-nucleoside DNA methylation inhibitors versus 5-aza-2'-deoxycytidine Mol. Cancer Ther., October 1, 2005; 4(10): 1515 - 1520. [Abstract] [Full Text] [PDF]
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 M. Z. Fang, D. Chen, Y. Sun, Z. Jin, J. K. Christman, and C. S. Yang Reversal of Hypermethylation and Reactivation of p16INK4a, RAR{beta}, and MGMT Genes by Genistein and Other Isoflavones from Soy Clin. Cancer Res., October 1, 2005; 11(19): 7033 - 7041. [Abstract] [Full Text] [PDF]
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 W. J. Lee, J.-Y. Shim, and B. T. Zhu Mechanisms for the Inhibition of DNA Methyltransferases by Tea Catechins and Bioflavonoids Mol. Pharmacol., October 1, 2005; 68(4): 1018 - 1030. [Abstract] [Full Text] [PDF]
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Z. Hou, S. Sang, H. You, M.-J. Lee, J. Hong, K.-V. Chin, and C. S. Yang Mechanism of Action of (-)-Epigallocatechin-3-Gallate: Auto-oxidation-Dependent Inactivation of Epidermal Growth Factor Receptor and Direct Effects on Growth Inhibition in Human Esophageal Cancer KYSE 150 Cells Cancer Res., September 1, 2005; 65(17): 8049 - 8056. [Abstract] [Full Text] [PDF]
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S. A. Belinsky Silencing of genes by promoter hypermethylation: key event in rodent and human lung cancer Carcinogenesis, September 1, 2005; 26(9): 1481 - 1487. [Abstract] [Full Text] [PDF]
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M. Kim, A. Murakami, K. Kawabata, and H. Ohigashi (-)-Epigallocatechin-3-gallate promotes pro-matrix metalloproteinase-7 production via activation of the JNK1/2 pathway in HT-29 human colorectal cancer cells Carcinogenesis, September 1, 2005; 26(9): 1553 - 1562. [Abstract] [Full Text] [PDF]
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B. Brueckner, R. Garcia Boy, P. Siedlecki, T. Musch, H. C. Kliem, P. Zielenkiewicz, S. Suhai, M. Wiessler, and F. Lyko Epigenetic Reactivation of Tumor Suppressor Genes by a Novel Small-Molecule Inhibitor of Human DNA Methyltransferases Cancer Res., July 15, 2005; 65(14): 6305 - 6311. [Abstract] [Full Text] [PDF]
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M. Fenech The Genome Health Clinic and Genome Health Nutrigenomics concepts: diagnosis and nutritional treatment of genome and epigenome damage on an individual basis Mutagenesis, July 1, 2005; 20(4): 255 - 269. [Abstract] [Full Text] [PDF]
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 K. Miyamoto and T. Ushijima Diagnostic and Therapeutic Applications of Epigenetics Jpn. J. Clin. Oncol., June 1, 2005; 35(6): 293 - 301. [Abstract] [Full Text] [PDF]
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 J. W. FINLEY Proposed Criteria for Assessing the Efficacy of Cancer Reduction by Plant Foods Enriched in Carotenoids, Glucosinolates, Polyphenols and Selenocompounds Ann. Bot., June 1, 2005; 95(7): 1075 - 1096. [Abstract] [Full Text] [PDF]
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P. W. Laird Cancer epigenetics Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R65 - R76. [Abstract] [Full Text] [PDF]
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X.-c. Xu, J. J. Lee, T.-T. Wu, A. Hoque, J. A. Ajani, and S. M. Lippman Increased Retinoic Acid Receptor-{beta}4 Correlates In vivo with Reduced Retinoic Acid Receptor-{beta}2 in Esophageal Squamous Cell Carcinoma Cancer Epidemiol. Biomarkers Prev., April 1, 2005; 14(4): 826 - 829. [Abstract] [Full Text] [PDF]
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M. Shimizu, A. Deguchi, J. T.E. Lim, H. Moriwaki, L. Kopelovich, and I. B. Weinstein (-)-Epigallocatechin Gallate and Polyphenon E Inhibit Growth and Activation of the Epidermal Growth Factor Receptor and Human Epidermal Growth Factor Receptor-2 Signaling Pathways in Human Colon Cancer Cells Clin. Cancer Res., April 1, 2005; 11(7): 2735 - 2746. [Abstract] [Full Text] [PDF]
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Q.-Y. Lu, Y.-S. Jin, A. Pantuck, Z.-F. Zhang, D. Heber, A. Belldegrun, M. Brooks, R. Figlin, and J. Rao Green Tea Extract Modulates Actin Remodeling via Rho Activity in an In vitro Multistep Carcinogenic Model Clin. Cancer Res., February 15, 2005; 11(4): 1675 - 1683. [Abstract] [Full Text] [PDF]
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L.-C. Li, P. R. Carroll, and R. Dahiya Epigenetic Changes in Prostate Cancer: Implication for Diagnosis and Treatment J Natl Cancer Inst, January 19, 2005; 97(2): 103 - 115. [Abstract] [Full Text] [PDF]
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H. Matsubayashi, N. Sato, K. Brune, A. L. Blackford, R. H. Hruban, M. Canto, C. J. Yeo, and M. Goggins Age- and Disease-Related Methylation of Multiple Genes in Nonneoplastic Duodenum and in Duodenal Juice Clin. Cancer Res., January 15, 2005; 11(2): 573 - 583. [Abstract] [Full Text] [PDF]
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Y. Yuasa, H. Nagasaki, Y. Akiyama, H. Sakai, T. Nakajima, Y. Ohkura, T. Takizawa, M. Koike, M. Tani, T. Iwai, et al. Relationship between CDX2 gene methylation and dietary factors in gastric cancer patients Carcinogenesis, January 1, 2005; 26(1): 193 - 200. [Abstract] [Full Text] [PDF]
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S. Chen, M. Cho, K. Karlsberg, D. Zhou, and Y.-C. Yuan Biochemical and Biological Characterization of a Novel Anti-aromatase Coumarin Derivative J. Biol. Chem., November 12, 2004; 279(46): 48071 - 48078. [Abstract] [Full Text] [PDF]
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 C. D. Davis and E. O. Uthus DNA Methylation, Cancer Susceptibility, and Nutrient Interactions Experimental Biology and Medicine, November 1, 2004; 229(10): 988 - 995. [Abstract] [Full Text] [PDF]
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 A. F. List, J. Vardiman, J.-P. J. Issa, and T. M. DeWitte Myelodysplastic Syndromes Hematology, January 1, 2004; 2004(1): 297 - 317. [Abstract] [Full Text] [PDF]
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