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,***,3
,#,***,##4
*
Departments of Biochemistry,
Pathology,
Child Health,
#
Animal Sciences,
**
Nutrition and
Veterinary Pathobiology,
***
Genetics and
##
Molecular Biology Programs, University of Missouri, Columbia, MO 65211, and
Department of Biology, City University of New York, NY 11225
4To whom correspondence should be addressed. E-mail: lubahnd{at}missouri.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • genistein • phytoestrogen • DNA methylation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). These studies are based on consumption studies of phytoestrogen-rich diets. The causal relationship and the mechanisms of phytoestrogen action have yet to be determined (4
). Genistein, one of the many phytoestrogens contained in soy, has been shown to inhibit the proliferation of both breast and prostate cancer cell lines (5
). Genistein (Fig. 1
) has also been shown to compete with estradiol and play a role in cell growth through a pathway mediated by estrogen receptor (6
).
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–11
). Approximately 200,000 new cases of prostate cancer are diagnosed each year; approximately 45,000 men die from the disease. The high levels of soy consumed in Asian countries are thought to be one factor that may be responsible for this discrepancy. Indeed, genistein in soy diets has been shown to reduce the incidence of poorly differentiated prostatic adenocarcinoma in the transgenic adenocarcinoma of the mouse prostate (TRAMP5
) mouse model (12
) and our own unpublished work. The molecular action of genistein in the model is unknown, but we hypothesize that estrogens and genistein work through an estrogen receptor (ER) (4
) pathway to induce epigenetic mutations in DNA sequence (13
) and thus alter transcriptional activity.
Gene regulation occurs at many levels along the transcription-translation pathway. At the DNA sequence level, this regulation occurs either by base modification via mutation or by epigenetic regulation through mechanisms such as DNA methylation. These so-called epigenetic mutations in turn then serve to alter the DNA-histone protein interaction and subsequent chromatin structure, in a sense compacting chromatin so that transcriptional machinery cannot access gene promoter regions. DNA methylation has been shown to be associated with histone acetylation, which ultimately influences chromatin secondary structure and, in turn, gene expression (14
).
DNA methylation occurs by the covalent addition of methyl groups to the 5-position of cytosines that are 5' to guanines in the DNA sequence. Cytosine guanine (CpG) dinucleotides are found at only 
10% of their expected frequency in the genome, except in regions known as CpG islands, where they are found at or near the predicted frequencies (15
,16
). However, under normal circumstances, CpG islands are protected from methylation. These CpG islands are frequently located in gene promoter regions and therefore can serve in the regulation of gene expression. In a methylated promoter, transcription is shut down and therefore expression is silenced. Hypermethylation of genes classified as tumor suppressors or growth regulators is often deleterious to normal cell function and often results in abnormal growth and differentiation. Therefore, an assay designed to identify altered methylation patterns in the cancer genome would be highly beneficial in the field of cancer diagnostics. We have developed a technique for mice called mouse differential methylation hybridization (mDMH) to study the global methylation patterns in the mouse genome (17
,18
). This assay allows one to screen the entire genome for aberrant methylation patterns and locate candidate sequences for further study. Comparisons can therefore eventually be made between the methylation patterns of normal and diseased tissue.
Estrogenic compounds have been widely known to influence many stages of cancer development, but the mechanisms of these actions are not completely known. There have been many instances of both induction and protection from malignancies by estrogenic compounds. It is thought that exposure to plant estrogens may protect against certain types of malignancies; however, the mechanism of this protection is not known. We hypothesize that these phytoestrogens in some way influence changes in DNA methylation patterns and, therefore, gene expression. Using the mDMH technique, we have been able to show that phytoestrogens do indeed influence DNA methylation in C57BL/6J mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Diet.
Mice were maintained on casein-based diet American Institute of Nutrition (AIN) 93G. Diet composition included: high-nitrogen casein (ICN Biomedicals, Aurora, OH); corn starch (National Starch and Chemical Co, Bridgewater, NJ); dextrose (Dyetrose; Dyets, Bethlehem, PA); sucrose and corn oil (Allen Foods, St Louis, MO); cellulose (Alphacell, ICN Biomedicals); safflower oil, choline bitartrate and DL-methionine (ICN Biomedicals); mineral mix (AIN 93G; ICN Biomedicals); vitamin mix (AIN 93VX; ICN Biomedicals) and 300 mg genistein/kg diet (LC Labs, Woburn, MA). See Table 1
for dietary concentrations. The number of mice was selected for convenience and availability. Amplicons were generated from each mouse and screened on three membranes each, totaling 900 CpG clones.
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). Tissue samples were then collected from brain, kidney, liver, spleen, prostate and testes and then flash frozen in liquid nitrogen. Mice were maintained on a daily light/dark cycle of 12 h:12 h, with ambient temperature and humidity set at 21°C and 50%, respectively.
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8.0] and Proteinase K (250 µg/mL). The digested tissue was then extracted twice with an equal volume of 5.24:1 phenol/chloroform/isoamyl alcohol. The aqueous phase was then extracted again with 24:1 chloroform/isoamyl alcohol. The DNA was then precipitated using 0.2 M NaAC and 2 volumes of 100% ethanol (EtOH). The DNA pellet was washed twice in 70% EtOH and resuspended in 100–200 µL of 1x TE (10 mM Tris, 0.2 M EDTA).
Amplicon generation.
DMH was described previously (17
,19
). Amplicons were generated by first digesting 1 µg of mouse prostate DNA with the restriction enzyme MseI [T/TAA; New England Biolabs (NEB), Beverly, MA]. Digestion reactions followed the specifications outlined by the supplier. DNA was ligated to double-stranded PCR linkers generated from single-stranded oligomers (H-24, 5'-AGG CAA CTG TGC TAT CCG AGG GAT-3'; H-12, 5'-TAA TCC CTC GGA-3') that were combined and cooled from 65°C to room temperature. DNA was then digested with BstUI cuts unmethylated (*CG/*CG) or HpaII cuts unmethylated (*CC/GG) (NEB); both enzymes are inhibited if cytosines within the CpG dinucleotide are methylated within the restriction site. As a control, samples were also cut with MspI (NEB), a methylation-insensitive isoschizomer of HpaII. After digestion, samples were PCR-amplified using primers specific for the ligated linkers (H-24). Amplicon products were labeled with [32P]dCTP using the Megaprime labeling system (Amersham Biosciences, Piscataway, NY) and hybridized to DMH array nylon membranes overnight at 65°C using the high-efficiency hybridization system (HEHS; Molecular Research, Cincinnati, OH). Membrane washing was done by first using a low-stringency/high-salt wash of 0.1% SDS, 0.5x SSC (0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) for 25 min and then a high-stringency/low-salt wash (0.01% SDS, 0.2x SSC) three times with 25 min for each wash. Autoradiographic exposure varied depending on the intensity of exposure desired. Sequence analysis was performed in the Missouri University DNA Core, and resulting sequences were compared with known sequence by Basic Local Alignment Search Tool (BLAST) analysis.
Membrane array generation
Candidate mouse CpG island clones from a mouse CpG island library [CGIL; United Kingdom Human Genome Mapping Project (UK-HGMP), Hinxton, Cambridge, United Kingdom] were cultured in 96-well plates and used as template DNA to generate PCR product plates containing amplified CpG island inserts to be blotted onto nylon membranes using a 96-pin MULTI-PRINT replicator (V&P Scientific, San Diego, CA). The nylon arrays were then hybridized with 32P-labeled amplicons overnight at 65–70°C (described above). Differentially methylated clones were then sequenced using the automated DNA sequencing protocol from the University of Missouri-Columbia DNA core.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Table 3
). All of these differences were seen in mice finishing the 4 continuous weeks of genistein diet. One clone of interest (4MC2-C8, shown on Figure 2
, named by location in the 96-well culture plates used to grow the library) was chosen for further study. This clone was chosen for its variation in intensity throughout the treatment groups, showing a consistent trend between animals. The other two clones showing methylation changes did not exhibit so clear a trend and will be studied further. After sequencing, it was determined that the clone was a CpG island found in a novel gene (Table 3)
. The duplicate spots on the 4-wk genistein membrane were significantly darker than the equivalent spots on any other membrane. This would suggest hypermethylation caused by genistein. The arrays screened with prostate DNA from mice fed only casein control diet showed lighter intensity from the 4MC2-C8 clones, whereas the arrays from the other two schemes containing 2 wk of genistein treatment showed an intensity between the total casein and total genistein. The 4MC2-C8 clones were very similar in intensity between the mice that had been fed genistein in the first 2 wk only, and those that had been fed genistein only in the last 2 wk. This methylation difference is currently being verified by Southern blotting. Preliminary blots do indicate hypermethylation of this clone (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). For many years, soy compounds have been thought to have protective effects against cancer, although the exact mechanism of action is not known. Their protective actions are most likely made up of not one but many different pathways. We have shown a difference in prostate DNA methylation patterns between mice treated with genistein and control; however, further studies are necessary to determine the significance of this finding and the role of methylated DNA in the progression of mouse prostate cancer.
Several reasons exist that may explain the differences in intensity of the spotted clones in Figure 2
. The first is the observation of changes in methylation in the identified genes after exposure to genistein. Another plausible explanation is a disproportionate difference in methylation patterns between different cell types. The reproductive tract consists of several cell types that have long been known to have different hormonal interactions as seen in different tissue recombinant studies (21
–23
). An idea is that DNA methylation patterns are caused by hormonal interactions, and methylation differences observed in our assay may be a result of varying quantities of DNA from heterogeneous mixtures of cells in these tissues. Because of the potential for these unequal mixtures and the use of PCR for generating amplicons, it is possible that variations in intensity may be a direct result of higher concentration of DNA from a particular cell type at the expense of the others. Also, tissue specific DNA methylation changes have been shown to exist (24
,25
), and with the rather crude method of tissue collection at our disposal, it is impossible to guarantee the homogeneity of the tissue sample. These could be possible explanations for some methylation patterns, but the use of duplicate mice on each diet scheme would tend to rule this out. The arrays from the two mice on each scheme were virtually identical, so this would indicate a unique change caused by our variable factor, diet.
This study indicates a partial change in the DNA methylation patterns of the mouse prostate after genistein exposure, but not in the mouse liver. Until recently, methylation studies have taken a candidate gene approach. In this assay, the presence or absence of a spot would indicate hypermethylation or hypomethylation, respectively, relative to the control. However, we have shown that intensity differences may also indicate a partial alteration in methylation status. We have shown that the intensity of the 4MC2-C8 spots on our arrays increases with the amount of time spent on the genistein diet. The arrays from the animals fed casein for 2 wk and then genistein for 2 wk and the animals fed genistein for 2 wk and then casein for 2 wk were virtually identical, suggesting that the particular time of treatment may be relevant compared with those that were on genistein for the full 4 wk. It suggests also that the methylation patterns were somewhat stable over a period of 2 wk and by 4 wk may become less stable. Expression analysis is necessary to determine the exact significance of the methylation changes.
This study supports the reproducibility of the mDMH assay and reasserts its value in screening global DNA methylation changes. We have shown that the screening of the library arrays with amplicons generated with DNA from different mice show a high degree of similarity. We have found that the amount of DNA used throughout the amplicon generation process is very important in the results (data not shown). The amount of DNA must be constant throughout because of the sensitive nature of PCR, or false positives may result. To correct for this, we have instituted several steps of DNA quantitation before each PCR step in the amplicon protocol.
The mDMH assay allows for the quick and easy identification of mouse CpG island target sequences, which can be amplified and sequenced for further analysis. All clones that show differential methylation on the arrays can be verified with Southern blotting or sodium bisulfite sequencing. We are concentrating our efforts now on identifying more target sequences and studying the relationship between other phytoestrogens and the progression of prostate cancer in the TRAMP mouse model. Additional studies to evaluate differential dosage and time-course effects of genistein and other phytoestrogens on DNA methylation are underway or planned.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 This work was supported by the Missouri University Center for Phytonutrient and Phytochemical Studies and National Institutes of Health grants PO1-ES10535 and R25-GM56901-04.
3 Current address: Department of Molecular Biology, Epigenomics, Inc., Seattle, WA 98101.
5 Abbreviations used: AIN, American Institute of Nutrition; BLAST, Basic Local Alignment Search Tool; CGIL, cytosine guanine dinucleotide island library; CpG, cytosine guanine dinucleotide; D44443, dexamethasone-induced product gene; DMH, differential methylation hybridization; ER, estrogen receptor; EtOH, ethanol; HEHS, high-efficiency hybridization system; mDMH, mouse differential methylation hybridization; NEB, New England Biolabs; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, standard saline citrate; TE, Tris/EDTA; TRAMP, transgenic adenocarcinoma of the mouse prostate; UK-HGMP, United Kingdom Human Genome Mapping Project.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Adlercreutz, H. (1995) Phytoestrogens: epidemiology and a possible role in cancer protection. Environ. Health Perspect. 103:103-112.
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18. Huang, T. H.-M., Laux, D. E., Hamlin, B. C., Tran, P., Tran, H. & Lubahn, D. B. (1997) Identification of DNA methylation markers for human breast carcinomas using the methylation-sensitive restriction fingerprinting technique. Cancer Res 57:1030-1034.
19. Yan, P. S., Perry, M. R., Laux, D. E., Asare, A. L., Caldwell, C. W. & Huang, T. H. (2000) CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer. Clin. Cancer Res. 6:1432-1438.
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22. Cunha, G. R., Young, P., Hom, Y. K., Cooke, P. S., Taylor, J. A. & Lubahn, D. B. (1997) Elucidation of a role for stromal steroid hormone receptors in mammary gland growth and development using tissue recombinants. J. Mammary Gland Biol. Neoplasia 2:393-402.[Medline]
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and ERß. ER