ToxSci Advance Access originally published online on December 16, 2006
Toxicological Sciences 2007 96(1):72-82; doi:10.1093/toxsci/kfl188
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Orphan Nuclear Receptor Constitutive Active/Androstane Receptor–Mediated Alterations in DNA Methylation during Phenobarbital Promotion of Liver Tumorigenesis
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* Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824; Laboratories of
Reproductive and Developmental Toxicology and
Experimental Pathology, NIEHS, NIH, Research Triangle Park, North Carolina 27709
Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824
1 To whom correspondence should be addressed at Department of Pharmacology and Toxicology, Michigan State University, B-440 Life Sciences Bldg., East Lansing, MI 48824. Fax: (517) 353-8915. E-mail: goodman3{at}msu.edu.
Received October 26, 2006; accepted December 12, 2006
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ABSTRACT |
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Altered DNA methylation is an epigenetic mechanism that plays a key role in the carcinogenesis process, and the nongenotoxic rodent hepatocarcinogen phenobarbital (PB) alters the methylation status of DNA in mouse liver. The constitutive active/androstane nuclear receptor (CAR) mediates half of the PB-induced hepatic gene expression changes and it is essential for liver tumor promotion in PB-treated mice. Here, a technique involving methylation-sensitive restriction digestion, arbitrarily primed PCR, and capillary electrophoresis was utilized to detect PB-induced regions of altered DNA methylation (RAMs) in CAR wildtype (WT) mice that are sensitive to promotion by PB and resistant CAR knockout (KO) mice. The CAR WT mice developed preneoplastic lesions after 23 weeks of PB treatment (precancerous) and liver tumors after 32 weeks, while the CAR KO mice did not develop tumors (Y. Yamamoto, et al., 2004, Cancer Res. 64, 7197–7200). Our goal was to discern those RAMs which are playing important roles in tumor formation by comparing the RAMs that form in sensitive and resistant groups of mice. Using this novel approach, 42 unique RAMs were identified in the precancerous as compared to the CAR KO, 23-week PB-treated tissue. Of these 42 RAMs, 14 carried forward to the tumor tissue, and additionally, 104 total unique RAMs were observed in the tumor tissue. These results indicate that there are unique RAMs occurring in the sensitive CAR WT mice and that a portion of these are seen in both the precancerous and tumor tissue. We hypothesize that these unique RAMs may be facilitating the tumorigenesis process, and these data support the view that DNA methylation plays a causative role in PB-induced tumorigenesis.
Key Words: CAR; DNA methylation; mouse liver; phenobarbital; tumors.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTAL DATAREFERENCES |
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The constitutive active/androstane receptor (CAR) is a member of the orphan nuclear receptor subfamily, which lacks known physiological ligands (Mohan and Heyman, 2003
). CAR is expressed primarily in the liver and the small intestine, and at lower levels in heart, muscle, kidney, and lung tissue (Baes et al., 1994). The role of CAR in detoxification is supported by the observations that it is activated by a variety of xenobiotics and mediates the upregulation of several phase I and II drug-metabolizing enzymes, for example, CYP450s, glutathione S-transferases and uridine diphosphate-glucuronosyltransferases (Ueda et al., 2002a) in addition to drug transporters (Assem et al., 2004; Kast et al., 2002). CAR also contributes to the metabolism of endogenous compounds such as bilirubin, and steroid and thyroid hormones in response to exogenous agents (Kodama and Negishi, 2006), and its regulation of enzymes involved in energy metabolism is a component of the response to xenobiotic stress (Swales and Negishi, 2004). Finally, in addition to acting as a component of a transcription factor, CAR may interact with proteins leading to alteration of their function, for example, CAR binding to the FoxO1 protein inhibits the ability of the latter to increase gluconeogenesis (Kodama and Negishi, 2006).
Phenobarbital (PB) is a prototypical nongenotoxic rodent liver tumor promoter that stimulates the expression of drug-metabolizing enzymes, in addition to causing hepatic hypertrophy and hyperplasia (Whysner et al., 1996). PB facilitates the translocation of CAR from the cytoplasm to the nucleus where it forms a heterodimer with the retinoid X receptor which can bind to the PB-responsive element located within the promoter region of particular genes, for example, Cyp2b10, leading to upregulation of their expression (Honkakoski et al., 1998). Furthermore, microarray data from Ueda et al. (2002a) indicate that PB alters the expression of 138 hepatic genes, increasing the expression of some while decreasing the expression of others, and approximately half of these changes are CAR dependent. Thus, not all of the hepatic effects of PB are CAR-dependent. CAR also mediates PB-induced hepatomegaly (Wei et al., 2000). CAR is essential for liver tumor promotion by PB in C3H/He mice following initiation with diethylnitrosamine (DEN) (Yamamoto et al., 2004). Additionally, chronic activation of CAR by the potent PB-like inducer 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) results in mouse liver tumor formation (Huang et al., 2005).
Altered DNA methylation, an epigenetic mechanism which plays a key role in the regulation of gene expression, may play multiple roles in carcinogenesis (Counts and Goodman, 1995). The epigenetic DNA modification 5-methylcytosine accounts for approximately 3–5% of all DNA bases and is found at 70–80% of all CpG dinucleotides in the genome (Ehrlich et al., 1982). Methylation at CpNpG sites also occurs in the mammalian genome (Clark et al., 1995). Aberrant patterns of DNA methylation have been implicated in carcinogenesis. For example, the genomes of cancer cells are globally hypomethylated as compared to their normal counterparts (Gama-Sosa et al., 1983) and decreased levels of methylation have been shown to cause activation of oncogenes such as R-ras and cyclin D2 (Nishigaki et al., 2005; Oshimo et al., 2003), in addition to chromosomal instability and increased tumor formation (Eden et al., 2003; Gaudet et al., 2003). Concurrently, GC-rich regions in the promoters of tumor suppressor genes in cancer cells become hypermethylated, resulting in gene silencing (Jones and Laird, 1999). Both mutation and epigenetic alterations are thought to play critical roles at all stages of the multistep/multistage model of the carcinogenesis process (Watson and Goodman, 2002), which can be divided into experimentally determined stages, that is, initiation, promotion, and progression (Dragan et al., 1993; Pitot, 1990).
Tumor promoters increase cell proliferation in target tissue and suppress apoptosis, thus facilitating the clonal expansion of initiated cells (Schulte-Hermann et al., 1990). PB treatment alters hepatic DNA methylation status, and the effects, specifically hypermethylation of internal and external cytosines of 5' CCGG 3' sequences, are greater in liver tumor-prone as compared to resistant strains of mice (Watson and Goodman, 2002). In addition, unique PB-induced regions of altered methylation (RAMs) were identified in sensitive B6C3F1 mice as compared to relatively resistant C57BL/6 mice (Bachman et al., 2006b). The comparison of RAMs that form in liver of tumor-susceptible mice as compared to those that are relatively resistant results in the identification of RAMs that occur uniquely in the sensitive group and provides the opportunity to discern PB-induced methylation changes which may be crucial to the tumorigenesis process (Bachman et al., 2006b).
In the current study, the methylation status of DNA was evaluated in mouse liver samples used by Yamamoto et al. (2004) where CAR wildtype (WT) and CAR knockout (KO) animals received one initiating dose of DEN followed by treatment with PB. CAR WT mice developed preneoplastic hepatic lesions after 23 weeks and tumors after 32 weeks of PB treatment, while the CAR KO mice did not form preneoplastic hepatic lesions after 23 weeks of PB treatment and liver tumors were not observed after 32 weeks of PB treatment (Yamamoto et al., 2004). The PB-induced RAMs that occurred in the sensitive CAR WT mice were compared to those in the resistant CAR KO mice in order to evaluate the hypothesis that the RAMs that formed uniquely in the sensitive mice may play a role in key events that occur during liver tumor formation. Our goal is to identify RAMs that play a role in mouse liver tumorigenesis by ascertaining those that occur uniquely in the sensitive CAR WT mice as compared to resistant CAR KO mice.
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MATERIALS AND METHODS |
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Animals, Treatments, and Tissue Samples
CAR KO (homozygous null) mice on a C57BL/6 background were backcrossed to C3H/HeNCrlBR mice (Charles River Laboratories, Wilmington, MA) (Yamamoto et al., 2004
). The C3H/He mouse is a strain that is highly susceptible to liver tumorigenesis (Buchmann et al., 1991). The male mice used for these studies were housed in a specific pathogen-free facility and cared for in accordance with the National Institute of Health guidelines for the care and use of laboratory animals. CAR WT or CAR KO mice were injected with a single intraperitoneal dose of DEN, 90 mg/kg, at 5 weeks of age and then administered drinking water (control) or 0.05% PB (w/w) in drinking water starting at 7 weeks of age and continuing for 23 or 32 weeks, resulting in the following groups of mice: CAR KO, 23 weeks control; CAR KO, 23 weeks PB; CAR WT, 23 weeks control; CAR WT, 23 weeks PB (precancerous tissue); and CAR WT, 32 weeks PB (tumor tissue) (Yamamoto et al., 2004). Animals were sacrificed at the end of the indicated time points, and their livers were quickly removed and frozen in liquid nitrogen. Liver samples were obtained from six mice in each experimental group, tissue samples from the CAR WT, 23-week PB group were taken from nonlesion areas of the liver, and the six samples of tumor tissue were obtained. Histopathology was performed. Tumors were selected based upon a limited availability for methylation studies, four adenomas and two carcinomas were selected. Thus, we were able to evaluate control tissue versus precancerous (CAR WT, 23 weeks PB) and precancerous tissue versus tumor tissue. However, due to the limited availability of samples, we were not able to evaluate specifically adenomas versus carcinomas. Regarding the precancerous tissue (CAR WT, 23 weeks PB), there were no visible alterations, that is, no nodules and no adenomas (based upon histology of adjacent tissue). However, there could have been microscopic foci of cellular alteration in the frozen samples. Overall, the adjacent fixed specimens did occasionally have one or two small foci.
DNA isolation.
Liver tissue was homogenized in 1 ml of TRIzol Reagent (Invitrogen, Carlsbad, CA). DNA was isolated via the manufacturer's protocol.
Evaluation of DNA Methylation Status by Arbitrarily Primed PCR and Capillary Electrophoresis
DNA methylation status was evaluated by an arbitrarily primed PCR (AP-PCR) and capillary electrophoresis procedure (Bachman et al., 2006b). This technique permits the simultaneous evaluation of treatment-related hypomethylations (less methylation in a region that was methylated in control), hypermethylations (more methylation in a region that was methylated in control), and new methylations (methylation in a region that was not methylated in control) in numerous regions of the genome.
Restriction digests.
Each DNA sample was subjected to three separate double restriction digests that were performed in duplicate with (1) a methylation-insensitive enzyme and (2) a methylation-sensitive enzyme. Digestion with a methylation-sensitive enzyme, RsaI or BfaI, ensures complete digestion by the methylation-sensitive enzyme, MspI, HpaII, or BssHII.
Restriction digestion with RsaI/MspI and RsaI/HpaII was carried out as described previously (Bachman et al., 2006b). MspI and HpaII recognize 5' CCGG 3' sites and cut between the internal cytosine and guanine. MspI will not restrict DNA if the external cytosine is methylated, while HpaII will not restrict DNA if the internal cytosine is methylated.
Restriction digestion with BfaI/BssHII was performed as described in a previous study (Bachman et al., 2006b). BssHII recognizes 5' GCGCGC 3' sites and cuts between the first guanine and cytosine residues and will not restrict DNA if several combinations of cytosines in the sequence are methylated (http://rebase.neb.com/cgi-bin/msget?BssHII). BfaI sites appear less frequently in CpG islands than RsaI sites and, thus, CpG islands are more intact following digestion of DNA with the former enzyme as compared to the latter (Shiraishi et al., 1995). Therefore, the use of BfaI in combination with BssHII, a 6-base rare cutter with a GC-rich recognition sequence, provides an additional, important "window" into our DNA methylation status analysis which complements the data obtained from the RsaI/MspI and RsaI/HpaII digests.
AP-PCR and Capillary Electrophoretic Separation of Products
AP-PCR and capillary electrophoresis were performed as described previously (Bachman et al., 2006b), however, two corrections were made: The five cycle stage of the AP-PCR reaction was followed by 30 cycles (not 40 cycles stated originally, as this was an error) of 94°C for 30 s (not 15 s stated originally, as this was an error), 55°C for 15 s, and 72°C for 1 min. The digested DNA was subjected to AP-PCR using a single arbitrary primer, 5' AAC CCT CAC CCT AAC CCC GG 3' (Gonzalgo et al., 1997), which was fluorescently labeled at the 5' end with HEX (Integrated DNA Technologies, Coralville, IA). This primer binds well to GC-rich regions and the 5' CCGG 3' sequence at its 3' end increases the probability of binding to the MspI and HpaII restriction sites, permitting the detection of methylation at the site of primer annealing and between sites of primer annealing. AP-PCR products were quantified using the ND-1000 ultraviolet-visible Spectrophotometer (Nanodrop Technologies, Wilmington, DE).
Data analysis.
The results as size of PCR products, in base pairs, and their corresponding peak areas are analyzed using the Excel program. A consensus, average peak area is calculated for each experimental group. This permits the consensus peak areas of one group to be compared with another to determine if there are differences in methylation at a particular PCR product size, and if so, these PCR products are considered to be RAMs. In this study, (1) the CAR KO, 23-week PB data were compared to the CAR KO, 23-week control data, and (2) both the CAR WT, 23 weeks PB (precancerous tissue) and CAR WT, 32 weeks PB (tumor tissue) data were compared to the CAR WT, 23-week control data. Treatment-related RAMs include (1) hypomethylations, both 100% decreases and decreases which are statistically significant when compared to control, (2) hypermethylations which are increases that are statistically significant when compared to control, and (3) new methylations in which PCR product formed following treatment but did not form under control conditions. For each PCR product size, the consensus treated peak area was compared to the consensus control peak area using a Student's t-test (p < 0.05). Each statistically significant difference was counted as a change in methylation (categorized as hypo- or hypermethylations) and only those methylation changes that were statistically significant are reported. All 100% hypomethylations were considered significant. Additionally, all new methylations, indicated by the formation of PCR product in the treated group and no product formation in the control group at a particular PCR product size, were considered significant. A detailed description of the data analysis procedure was described previously (Bachman et al., 2006a,b).
Assumptions.
The following assumptions are made during the data analysis procedure: (1) Each separate PCR product of a defined size represents a distinct region of the genome, (2) a region can include one or more recognition sequences for the specific methylation-sensitive restriction enzyme employed located between the annealing sites of the up- and downstream primers; thus, the amount of each PCR product formed can be viewed as representing an "average" of the methylation status of the particular recognition sequences located between the up- and downstream primers, and (3) differences in the amount of each PCR product between the groups being compared represent the altered methylation status of a particular GC-rich region of DNA.
Statistical analysis.
In order to determine if the RAMs that occurred at the same PCR product size in two experimental groups are equivalent (common RAMs) or different (unique RAMs), analysis of variance (ANOVA) tests (p < 0.05) were performed. ANOVA tests analyze the difference between the means of two or more groups, and an advantage over the use of multiple t-tests is the decreased probability of finding significance by chance. One-way ANOVA analysis was utilized to compare the magnitudes of the methylation changes that occurred at the same PCR product size in the CAR WT, 23-week PB- (precancerous) and the CAR WT, 32-week PB-treated (tumor) tissue because only one independent variable (i.e., duration of PB treatment) exists. Two-way ANOVA tests were executed to determine if the magnitudes of the RAMs that occurred at the same PCR product size as a result of PB treatment in the CAR WT, 23 weeks and the CAR KO, 23-week groups were equivalent or different because there are two independent variables (i.e., duration of PB treatment and mouse strain).
Common and unique RAMs.
PCR products of identical size that exhibit methylation changes in two experimental groups (e.g., precancerous and CAR KO, 23-week PB-treated tissue or tumor and precancerous tissue) are considered to be common RAMs. A region is considered to be a carry forward RAM if the methylation changes associated with the common RAM in the precancerous and tumor tissue occur in the same direction and are equivalent as determined by one-way ANOVA (p < 0.05). RAMs in common between two experimental groups are considered to be unique RAMs if (1) the methylation changes are opposite in direction (i.e., in the same region, a hypomethylation is elicited by one treatment and a hypermethylation occurs as a result of a second treatment), or (2) the changes in methylation occur in the same direction (i.e., both changes are hypermethylations) but the extents of change are statistically different as determined by two-way, p < 0.05 (for precancerous vs. CAR KO, 23-week PB-treated tissue) or one-way ANOVA, p < 0.05 (for tumor vs. precancerous tissue). In addition, RAMs are considered to be unique if they are observed only in a particular treatment group. For instance, a RAM may be observed only in the precancerous tissue (vs. CAR KO, 23-week PB-treated tissue) or only in the tumor tissue (vs. precancerous tissue).
Percent Dissimilarity Calculations
Treatment group versus control group.
The dissimilarity between control patterns of methylation and PB-induced patterns was calculated for three treatment versus control groups (CAR KO, 23-week PB vs. CAR KO, 23-week control tissue, precancerous vs. CAR WT, 23-week control tissue, and tumor vs. CAR WT, 23-week control tissue). The total number of PCR products reporting in the control was added to the total number of unique PCR products (i.e., those PCR product sizes that were not formed under control conditions) reporting from treatment to obtain the total number of different sized PCR products reporting in the control and treatment groups. The total number of RAMs (hypomethylations, hypermethylations, and new methylations) in the treatment group divided by the total number of different sized PCR products that reported in the control and treatment group, expressed as a percent, equals the percent dissimilarity of the treatment as compared to the control pattern of methylation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTAL DATAREFERENCES |
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The procedure employed, which involves methylation-sensitive restriction digestion, AP-PCR, and capillary electrophoresis, permits the simultaneous detection of treatment-related hypermethylations, hypomethylations, and "new" methylations throughout GC-rich regions of the genome. Administration of PB resulted in numerous RAMs in the livers of CAR WT mice treated for 23 weeks, CAR KO mice treated for 23 weeks and in liver tumors observed following treatment of CAR WT mice for 32 weeks (Table 1 and Figs. 1–3). The actual basic, underlying data for Table 1 and Figures 1–3 are located in the Supplemental Information.
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are depicted in (B). Hypomethylations 
, hypermethylations 
, new methylations 
, and those RAMs in common between precancerous and CAR KO, 23-week PB-treated tissue and which exhibit methylation changes that occur in opposite directions are represented 
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, represented in (B), are segregated into hypomethylations 
, and those RAMs in common between the tumor and precancerous tissue with methylation changes that occurred in opposite directions 
.Treatment of WT mice with PB for 23 weeks (precancerous tissue) and 32 weeks (tumor tissue) resulted in the detection 47 and 129 RAMs, respectively (Table 1). Considerable increases of both hypomethylations and "new" methylations occurred, displaying greater then 2.6- and 4.3-fold increases, respectively, from precancerous to tumor tissue (Table 1). While the total number of RAMs detected in the precancerous and the CAR KO, 23-week PB-treated tissue was approximately equal, the specific alterations differed. There were no hypermethylations observed in the CAR KO, 23-week PB-treated tissue, while 10 occurred in the precancerous tissue. Furthermore, approximately twice the number of hypomethylations occurred in the CAR KO, 23-week PB-treated tissue as in the precancerous tissue (Table 1).
In order to ascertain methylation changes that may play key roles in liver tumor formation in the CAR WT C3H/He mice, unique RAMs in the precancerous as compared to the CAR KO, 23-week PB-treated tissue were first identified (Fig. 1A). There were 42 unique changes that occurred in the precancerous as compared to the CAR KO, 23-week PB-treated tissue (Fig. 1B).
From this pool of unique regions in the precancerous tissue, carry forward RAMs were discerned. Carry forward RAMs are those regions that were considered to be unique in the precancerous as compared to the CAR KO, 23-week PB-treated tissue, were also observed in the tumor tissue, and whose methylation changes at a common RAM in the tumor and precancerous were equivalent as determined by one-way ANOVA, p < 0.05 (Fig. 2A). Of the 42 unique RAMs in the precancerous tissue as compared to the CAR KO, 23-week PB-treated tissue, 14 (33%) carried forward to the tumor tissue (Fig. 2B). These carry forward changes were primarily hypomethylations (71%) but two hypermethylations and two new methylations also were observed (Fig. 2B).
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from precancerous to tumor tissue are depicted in (B). These RAMs are divided into hypomethylations Additionally, the total unique RAMs in the tumor tissue were detected (Fig. 3A). Unique RAMs in the tumor tissue as compared to the precancerous tissue consist of common RAMs at which the methylation changes in the tumor tissue occur to a greater extent as determined by one-way ANOVA analysis (p < 0.05), common RAMs with opposite directional changes in the tumor tissue (i.e., at a common RAM, PB induces a hypomethylation in the tumor tissue and a hypermethylation in the precancerous tissue), and unique RAMs observed only in the tumor tissue. Finally, to obtain the total unique RAMs in the tumor tissue, common RAMs in the tumor and CAR KO, 23-week PB-treated tissue that exhibited equivalent methylation changes as determined by two-way ANOVA (p < 0.05) were subtracted out, as methylation changes that occur in the CAR KO animals are most likely not critical for liver tumorigenesis.
In total, 104 unique RAMs were observed in the tumor tissue (Fig. 3B). New methylations accounted for 63% (66/104) of these changes, while hypomethylations represented 34% (35/104) and hypermethylations only 3% (3/104) of the total unique RAMs in the tumor (Fig. 3B). Both the carry forward RAMs and the total unique RAMs in the tumor tissue may play critical mechanistic roles during the formation of liver tumors in the CAR WT C3H/He mice.
A diagram illustrating all of the data previously described is shown (Fig. 4). Unique RAMs in the precancerous as compared to the CAR KO, 23-week PB-treated tissue are depicted, and from these regions, carry forward changes from the precancerous to tumor tissue were determined. Finally, to obtain the total unique RAMs in the tumor tissue, three hypomethylations and two new methylations were subtracted out from the tallied unique RAMs in the tumor as compared to the precancerous tissue (Fig. 4). These five RAMs were unique in the tumor as compared to the precancerous tissue and also were common RAMs between the tumor and CAR KO, 23-week PB-treated tissue, with their associated methylation changes deemed to be equivalent by two-way ANOVA, p < 0.05 (Fig. 4). These RAMs have been excluded from the total unique RAMs in the tumor tissue because they are in common and equivalent between the precancerous mice and the CAR KO, 23-week PB-treated mice that do not develop liver tumors, and thus are probably not relevant to the tumorigenesis process. We understand that this a conservative approach and we recognize that the methylation changes that are equivalent in the CAR KO, 23-week PB-treated and tumor tissue may be necessary but not sufficient for tumor development. It is important to note that all four of the unique RAMs in the tumor as compared to the precancerous tissue in which the methylation changes occurred in opposite directions (Fig. 3B) were PB-elicited hypomethylations in the tumor tissue (as compared to an increase in methylation at the common region in the precancerous tissue) and are categorized as such (Fig. 4).
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. RAMs that carried forward in the CAR WT mice from precancerous to tumor (CAR WT, 32 weeks PB) tissue are depicted The calculation of percent dissimilarity allows for the evaluation of PB-induced patterns of altered methylation in a treatment group as compared to a control group. Dissimilarity between the precancerous and the CAR WT, 23-week control tissue was 47% but increased to 83% in the tumor tissue (Fig. 5). This observation indicates that the methylation pattern became progressively aberrant with prolonged PB treatment. Furthermore, the CAR KO, 23-week PB-treated tissue demonstrated 53% dissimilarity in its methylation pattern as compared to the CAR KO, 23-week control tissue (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTAL DATAREFERENCES |
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We have developed an approach that involves methylation-sensitive restriction digestion, AP-PCR, and capillary electrophoretic separation of the resulting PCR products which permits the detection of treatment-related changes in methylation, that is sites of hypo-, hyper-, and "new" methylation, that occur simultaneously throughout multiple regions of the genome (Bachman et al., 2006b
). DNA is subjected to a double digest with a nonmethylation-sensitive enzyme plus one that is methylation sensitive: RsaI/MspI, RsaI/HpaII, or BfaI/BssHII. By evaluating CCGG sites, we are assessing the methylation status of approximately 7.45% of the CpG dinucleotides within the mouse genome, 4.37% of which are located within unique sequences and 3.08% of which are found in transposable elements (Fazzari and Greally, 2004). A significant advantage of using MspI and HpaII is that the methylation status at CpCpG sites as well as CpG sites are evaluated, respectively, affording the opportunity to complementarily assess a wider spectrum of methylation alterations. Restriction with BfaI leaves CpG islands more intact than RsaI (Shiraishi et al., 1995) thus increasing the probability that subsequent digestion with the 6-base cutter BssHII will allow for the determination of the methylation status at sites within CpG islands. The combination of restriction digests employed permits a robust evaluation of methylation status along multiple regions of the genome.
The nongenotoxic rodent tumor promoter PB decreases global methylation levels to a greater extent in liver tumor-prone B6C3F1 as compared to resistant C57BL/6 mice (Counts et al., 1996). Furthermore, higher levels of hypermethylation occurred at the internal and external cytosines of the recognition sequence 5' CCGG 3' in mice that are more susceptible to liver tumorigenesis as compared to mice that are relatively resistant (C3H/He >> B6C3F1 > C57BL/6) (Watson and Goodman, 2002).
Using the aforementioned approach, we recently evaluated the effect of PB on the methylation status of DNA from B6C3F1 and C57BL/6 mice (Bachman et al., 2006b). This method is an improvement over the polyacrylamide gel-based procedure used by Watson and Goodman (2002) because the current capillary electrophoresis-based technique permits a much more precise separation and quantification of the PCR products. Unique RAMs in the sensitive B6C3F1 mice were identified (Bachman et al., 2006b). The primary advantage of comparing methylation changes that occur in sensitive versus resistant mice is that the RAMs which may be most important for tumorigenesis (i.e., unique in the sensitive mice) are segregated out from those which arise only in the resistant group. This approach is conservative in the sense that we may be excluding some RAMs that are necessary but not sufficient for tumor development by "discarding" those that are in common and equivalent between B6C3F1 and C57BL/6 mice.
In the current study, another sensitive versus resistant comparison was made between PB-induced RAMs that formed in the livers of sensitive CAR WT mice, which develop preneoplastic lesions after 23 weeks and liver tumors after 32 weeks of PB treatment, and the resistant CAR KO mice which do not develop liver lesions after 23 weeks of promotion or tumors after 32 weeks. RAMs were elicited in all three groups, indicating that PB induces methylation changes in these mice in both a CAR-dependent and CAR-independent manner (Table 1). These results are consistent with microarray data that show that approximately half of the PB-induced gene expression changes that occur in mouse liver are CAR dependent while the other half are CAR independent (Ueda et al., 2002a). Furthermore, chronic activation of CAR causes tumor formation in mice (Huang et al., 2005; Yamamoto et al., 2004). All of these results are consistent with the concept that PB induces CAR-dependent and CAR-independent alterations, including methylation changes, in mouse liver. We hypothesize that PB-induced RAMs that occur uniquely in the CAR WT mice, especially those which carry forward from the 23- to 32-week time points, may play key roles underlying promotion of liver tumorigenesis by PB.
More RAMs formed in the tumor as compared to the precancerous tissue, suggestive of a time-dependent relationship between duration of PB treatment and number of RAMs (Table 1). The unique RAMs observed in the precancerous and tumor tissue (Fig. 4) might facilitate tumorigenesis, especially those that carry forward from an early (precancerous) to a later (tumor) stage of tumorigenesis. It is important to recognize that the precancerous tissue is heterogenous, comprised of both nonfocal tissue and preneoplastic foci. By identifying methylation changes that carry forward from the precancerous to the tumor tissue, we are able to focus upon the crucial changes that most likely occur in the progression of preneoplastic lesions to tumors.
Moreover, there were 104 total unique RAMs that formed in the tumor tissue, and this number does not include the five RAMs that were in common and equivalent between the KO, 23-week PB-treated and the tumor tissue which were excluded in an effort to better focus upon the RAMs that were completely unique to the sensitive CAR WT mice (Fig. 3B). This observation supports the multistep/multistage model of tumorigenesis, during which there is a progressive accumulation of cells that have obtained heritable changes (i.e., genetic or epigenetic), which are critical for the selection and clonal expansion of initiated cells (Dragan et al., 1993).
In addition to the possible involvement of mutations in key genes, epigenetic events have been implicated in the tumorigenesis process (Feinberg et al., 2006; Watson and Goodman, 2002). Hypermethylation can downregulate the expression of tumor suppressor genes such as MLH1, VHL, and RB1 (Jones and Laird, 1999) and hypomethylation can activate oncogenes (Nishigaki et al., 2005; Oshimo et al., 2003) plus increase genetic instability (Eden et al., 2003) and tumor burden (Gaudet et al., 2003). Thus, the presence of unique RAMs in the precancerous and tumor tissue as compared to the CAR KO, 23-week PB-treated tissue, indicates that epigenetic alterations are involved in PB-promotion of mouse liver tumors.
CAR may mediate hypomethylation by blocking the methyltransferase enzymes from acting on DNA or recruiting proteins responsible for active demethylation (Bhattacharya et al., 1999; Jost et al., 2001; Zhu et al., 2000). CAR could also induce hypomethylation by engaging cytidine deaminases, such as those from the Aid/Apobec family (Morgan et al., 2004). Conversely, CAR may facilitate hypermethylation by recruiting methyltransferases, as in the case of GCNF and the resulting downregulation of Oct-3/4 expression during gastrulation (Sato et al., 2006). Additionally, CAR could mediate gene expression changes that affect enzymes involved in 1-carbon metabolism, thereby decreasing levels of methyl groups available for S-adenosylmethionine formation. Similarly, diets devoid of choline and methionine, compounds which serve as methyl-group donors for the DNA methyltransferase reaction, cause liver cancer in rodents (Ghoshal and Farber, 1984). CAR may also affect expression and/or activity of the DNA methyltransferases (Dnmts 1, 3a, 3b).
The human isoform of CAR (hCAR) (Baes et al., 1994) differs from its murine counterpart (mCAR) (Choi et al., 1997) and structural variations seem to be responsible for some of the different responses of hCAR as compared to mCAR (Ueda et al., 2002b). For instance, Kawamoto et al. (2000) found that estradiol and estrone activated mCAR but not hCAR. Additionally, mCAR and hCAR are both activated by PB (Huang et al., 2005) but the PB-like inducer TCPOBOP is an agonist for mCAR and not hCAR (Tzameli et al., 2000). Conversely, the xenobiotic 6-(4-chlorophenyl-imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime is an agonist for hCAR but does not affect mCAR (Maglich et al., 2003). There also are important species differences in terms of crosstalk between CAR and the xenobiotic-sensing pregnane X receptor (PXR). For example, TCPOBOP activates mCAR and hPXR but not hCAR or mPXR (Moore et al., 2000). Similarly, clotrimazole can induce hPXR and inhibit hCAR but it does not effect mCAR (Moore et al., 2000). Finally, androstanol serves as an inverse agonist for CAR, with its effects being greater in mCAR as compared to hCAR, and it also activates both mPXR and hPXR (Moore et al., 2000). Thus, there are important structural and regulatory differences between mCAR and hCAR. These distinctions might, in part, provide a mechanistic basis, possibly including differences in capacity to maintain normal patterns of DNA methylation, for the observation that PB, while being able to promote rodent liver tumors, is not a human carcinogen (Lamminpää et al., 2002).
The question remains, "Why are humans more resistant to the effects of PB?" Part of the answer may lie downstream of CAR, in terms of the genes that are induced/repressed, some of which may be deregulated by methylation changes. Many of the same tumor suppressors and oncogenes, such as pRB, p53, PTEN, and MLH1 (Herzig and Christofori, 2002), that are involved in a variety of human cancers are identical to those implicated in mouse tumorigenesis, and so while the same genes may be affected during human and mouse tumorigenesis, lesions may form only in mice due to differences in regulation of these genes, and altered methylation may be a component of this process. In general, humans are less susceptible to carcinogenesis as compared to rodents (Rangarajan and Weinberg, 2003). In this context, it should be noted that DNA methylation patterns are better preserved in human as compared to rodent cells in culture (reviewed in Goodman and Watson, 2002). Therefore, humans might be more comparable to the resistant KO mice in the sense that the methylation changes that occur in the CAR KO mice and any alterations in humans may not be as detrimental as those that arise in the CAR WT mice, and this could be a result of downstream differences in the RAMs caused by mCAR versus hCAR.
The approach that our laboratory recently developed affords us the opportunity to detect treatment-related alterations in methylation, including sites of hyper-, hypo-, and "new" methylations. PB-elicited unique RAMs were identified in liver tumor-susceptible CAR WT mice as compared to the resistant CAR KO mice, and these changes, including unique RAMs in the precancerous and tumor tissue and especially those that carry forward from the early to the later time point, may play key roles in tumor formation in these mice. Currently, the PCR products that represent these key RAMs are being cloned, sequenced, and annotated in an attempt to identify specific, novel genes that are involved in the tumorigenesis process due to altered methylation.
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SUPPLEMENTAL DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTAL DATAREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/. Supplementary data contain a series of figures which provide the actual, basic underlying data for Table 1 and Figures 1–3. These figures provide the data for RAMs whose methylation changes were (1) statistically significant (hypomethylations, hypermethylations), (2) complete 100% hypomethylations, and (3) new methylations. Data are depicted for the CAR KO, 23-week PB-treated, precancerous (CAR WT, 23-week PB-treated), and tumor (CAR WT, 32-week PB-treated) tissue.
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ACKNOWLEDGMENTS |
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J.M.P. is a predoctoral fellow supported by National Institutes of Health/National Institute of Environmental Health Sciences training grant No. T32-ES-7255. Research support, in the form of a gift, from the R.J. Reynolds Tobacco Company is acknowledged gratefully.
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  J. M. Phillips and J. I. Goodman Identification of Genes that May Play Critical Roles in Phenobarbital (PB)-Induced Liver Tumorigenesis due to Altered DNA Methylation Toxicol. Sci., July 1, 2008; 104(1): 86 - 99. [Abstract] [Full Text] [PDF]
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