Effects of Phenobarbital on DNA Methylation in GC-Rich Regions of Hepatic DNA from Mice That Exhibit Different Levels of Susceptibility to Liver Tumorigenesis

Rebecca E. Watson and Jay I. Goodman,1

Department of Pharmacology and Toxicology, Michigan State University, B440 Life Sciences Building, East Lansing, Michigan 48824

Received January 7, 2002; accepted March 6, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA methylation is an important epigenetic mechanism involved in transcriptional control and altered patterns of methylation may lead to the aberrant gene expression contributing to carcinogenesis. Three groups of mice were used in the current study: the relatively liver-tumor-sensitive C3H/He strain and B6C3F1 stock (C57BL/6xC3H/He), as well as the relatively resistant C57BL/6 strain. For a 2-week period, animals from each group were given drinking water containing a tumor-promoting dose of phenobarbital (PB), a nongenotoxic rodent carcinogen. Methylation-sensitive restriction digests using HpaII or MspI were followed by PCR amplification using an arbitrary primer or primer pair, binding preferentially to guanine and cytysine (GC)-rich regions of DNA, including CpG islands. This procedure allows for assessment of methylation at the internal and/or external cytosine of the 5`-CCGG-3` sites recognized by MspI and HpaII. Results with the single primer indicated marked differences in PB-induced hypermethylation at external and internal cytosines of 5`-CCGG-3` sites: C3H/He >> B6C3F1 > C57BL/6. Results with the arbitrary primer pair indicated PB-induced hypermethylation at the external cytosine of 5`-CCGG-3` site: B6C3F1 > C3H/He, and a low level of hypomethylation at internal and external cytosine sites in C57BL/6. Thus, there was a clear indication of more methylation changes in GC-rich regions of DNA, primarily hypermethylation, in the tumor-sensitive groups of mice in response to PB treatment. Therefore, this study supports our hypothesis that the capacity to maintain normal methylation patterns is related inversely to tumor susceptibility.

Key Words: B6C3F1; carcinogenesis; C3H/He; C57BL/6; CpG islands; DNA methylation; GC-rich regions; liver; non-CpG methylation; phenobarbital; susceptibility; tumorigenesis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA methylation plays a key role in the regulation of transcription. In the mammalian genome, approximately 3–5% of cytosine residues are present as 5-methylcytosine, which is often, but not exclusively, within CpG dinucleotides of both promoter and nonpromoter regions (Bird, 1992Go; Momparler and Bonenzi, 2000). CpG-rich stretches of DNA 200 bp or longer with a G + C content of 50% or greater are termed CpG islands (Gardiner-Garden and Frommer, 1987Go). These are commonly found at 5` flanking, promoter regions of genes (Robertson and Jones, 2000Go).

In general, the density of methylation is related inversely to gene expression (Laird and Jaenisch, 1994Go). This relationship is particularly commonplace in CpG islands at promoter regions, where DNA methylation may block transcription factors from accessing their cognate cis elements and/or indirectly suppress transcription through methylated DNA binding proteins that recruit histone deacetylases, leading to chromatin condensation and subsequent gene silencing (Jones et al., 1998Go; Nan et al., 1998Go). In addition, a large amount of CpG methylation at non-CpG island regions is found within foreign DNA elements. Methylation of these CpG dinucleotides is related inversely to the expression of parasitic transposons, and is believed to protect genomic integrity (Robertson and Wolffe, 2000Go). However, in certain nonpromoter regions of imprinted genes there is a direct correlation between increased methylation and gene expression. For instance, methylation of a specific nonpromoter region is required for expression of the tumor suppressor Igf2r gene on the maternal allele (Birger et al., 1999Go). Similarly, methylation of a differentially methylated nonpromoter region is necessary for paternal expression of Igf2 (Bell and Felsenfeld, 2000Go). Therefore, in examining changes in DNA methylation and how this may relate to gene expression it is important to consider alterations in the methylation status of guanine and cytosine (GC)-rich sequences in both promoter and nonpromoter regions of genes.

Methylation patterns in tumor tissues characteristically exhibit a decrease in global methylation accompanied by some increased methylation in selected regions of DNA (Robertson and Jones, 2000Go). More specifically, hypermethylation in promoter regions of tumor suppressors, associated with silencing of these genes, is a common finding in cancer (Lin et al., 2001Go). Hypermethylation and transcriptional silencing of the tumor suppressor p16 promoter region is seen in B6C3F1 lung adenocarcinomas and other types of cancers (Esteller et al., 2001aGo; Patel et al., 2000Go). Similarly, loss of pRb expression in pituitary adenocarcinomas has been shown to be associated with methylation of RB1 promoter regions (Simpson et al., 2000Go). Additionally, hypomethylation also plays an important role in carcinogenesis. Hypomethylation may facilitate aberrant gene expression of raf and other oncogenes normally silenced by methylation (Ray et al., 1994Go). Furthermore, hypomethylation could lead to a decrease in genomic integrity by reducing the methylation-mediated silencing of foreign genomic elements (Laird, 1997Go).

Consistent with the observation of global hypomethylation in tumor samples and precancerous lesions as compared to normal tissue (Gama-Sosa et al., 1983Go), studies in our laboratory have shown that hepatic DNA samples from mice that received a tumor promoting dose of the rodent carcinogen phenobarbital (PB) have lower levels of global methylation compared to controls. This difference is substantially more prominent in the relatively tumor-sensitive C3H/He and B6C3F1 (C57BL/6xC3H/He) mice than in the relatively tumor-resistant C57BL/6 strain (Counts et al., 1996Go).

In the current study we have extended our analysis of global PB-induced methylation change in these 3 groups of mice varying in tumor susceptibility to examine the status of methylation in selected regions of DNA, i.e., GC-rich sequences. Assessment of methylation status was performed using an arbitrarily primed PCR approach. The primers used were designed to bind to GC-rich sequences that are particularly prevalent at CpG islands (Gonzalgo et al., 1997Go). PB induced an increase in methylation in GC-rich regions that was more pronounced in the relatively tumor sensitive C3H/He and B6C3F1mice than in the relatively tumor resistant C57BL/6 strain. While our previous investigations indicated that global hypomethylation occurs as a result of PB treatment and the current study detects hypermethylation in GC-rich sequences, results are quite compatible and, indeed, complementary. The important point is that a variety of alterations in methylation may facilitate carcinogenesis and the methylation patterns of PB-treated C3H/He and B6C3F1 animals deviate more from their control counterparts than what is seen in the C57BL/6 strain.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Male C57BL/6, C3H/He, and B6C3F1 (C57BL/6xC3H/He) mice were obtained from Charles River Laboratories (Wilmington, MA). All animals were 43–63 days old, and weighed 22–24 g. Animals were housed in a temperature-controlled environment and given food and water ad libitum. Treatment animals were given a tumor-promoting dose of PB (0.05% w/w) in the drinking water for a 2-week period. Animals were sacrificed by CO2 asphyxiation, and their livers were snap frozen at –80°C.

DNA isolation and restriction digests.
DNA was extracted by a phenol/chloroform procedure (Strauss, 1990Go). For each DNA sample, 2 restriction digests were performed: 1 with RsaI and MspI and 1 with RsaI and HpaII. All enzymes used were from Boehringer-Mannheim. RsaI is methylation-insensitive, while MspI and HpaII are methylation-sensitive. Both MspI and HpaII cut between cytosine residues at 5`-CCGG-3` sites. MspI will not cut if the external cytosine is methylated, and HpaII will not cut if the internal cytosine is methylated (Mann and Smith, 1977Go). Restriction digests were performed with 1 µg of DNA and 5.0 units of RsaI in Boehringer-Mannheim buffer L. After a 1-h incubation with shaking in a water bath at 37°C, two 2.5 unit aliquots of MspI or HpaII were added, 2 h apart. The total incubation time was 18 h. The enzymes were inactivated by a 10-min incubation at 65°C and the digests were stored at 4°C until use.

Arbitrarily primed (AP)-33P PCR.
PCR was performed on restriction digests using a single arbitrary primer, 5`-AACCCTCACCCTAACCCCGG-3`, or a combination of arbitrary primers: 5`-AACCCTCACCCTAACCGCGC-3` and 5`-AACCCTCACCCTAACCCGCG-3` (Gonzalgo et al., 1997Go). While both the single primer and the primer pair were designed to bind CpG rich regions of DNA, they do not bind identical regions of DNA; thus, PCR products produced are distinct. Each PCR sample was prepared in a sterile laminar flow hood on ice with appropriate negative no DNA template controls. Reactions were composed of 10 µl of the restriction digest (containing 1 µg digested DNA), 0.4 mM each primer, 2.5 units of Taq polymerase (Gibco BRL), 1.5 mM MgCl2, 60 mM Tris, 15 mM ammonium sulfate, 3.3 µCi 33P (New England Nuclear), and glass-distilled water to volume. Samples were heated for 5 min at 94°C before addition of dNTPs to minimize the possibility of primer-dimer formation. Cycling conditions included a single denature cycle for 2 min at 94°C, followed by 5 cycles of the following conditions: 30 s at 94°C, 1 min at 40°C, 1.5 min at 72°C, then 40 cycles of 94°C for 30 s, 55°C for 15 s and 72° for 1 min, a time delay cycle for 5 min at 72°C, and a soak cycle at 4°C. The 40 cycle run was used in order to maximize the opportunity to amplify regions of interest. PCR products (5 µl from each reaction) were separated on a Stratagene Castaway Precast Sequencing Gel (6% polyacrylamide, 1X TBE, 7M urea) at 50W. The gel was soaked for 10 min in a fixing solution containing 5% acetic acid and 5% methanol, then rinsed for 10 min in glass-distilled water. The gels were dried and exposed to a Kodak phosphoimage screen. A short exposure of 2–5 days, followed by a longer exposure of 5–9 days was performed for each gel. Images from short and long exposures were analyzed separately. This procedure led to the separation of PCR products ranging from approximately 75–1500 bp. as verified by separating a labeled DNA marker on the same type of gel under identical conditions.

Analysis of phosphoimages.
Phosphoimages were analyzed with a Molecular Dynamics phosphoimager and Quantity One software (Bio-Rad). Banding patterns of 33P-PCR product phosphoimages were examined to determine the methylation status at external and internal cytosines at 5`-CCGG-3` sequences. Segments of DNA between or at sites of primer annealing are amplifiable by PCR unless a site within the region is cut with HpaII or MspI. Thus, bands seen in both MspI and HpaII digest lanes are indicative of the absence of unmethylated 5`-CCGG-3` sites. Bands present in HpaII digest lanes but not in MspI digest lanes represent methylation of the internal cytosine of a 5`CCGG 3` site. Conversely, bands seen more prominently in MspI digested lanes are indicative of methylation of the external cytosine. A hypothetical example is presented in Figure 1Go. We considered a control versus treatment group difference of 1–3, 4–6, and >6 bands seen in the MspI or HpaII lanes of 1 group that are either not present or seen less distinctly in the other as a small, moderate, and large amount of methylation change, respectively. Data from Quantity One were exported to Excel where the percent intensity/total intensity of the lanes were calculated and graphed using SPSS Sigma Plot 2000.



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FIG. 1. Illustration of an analysis of hypothetical results. The correlation between hypothetical bands seen in RsaI/MspI (M) and RsaI/HpaII (H) digest lanes and the methylation status of a 5`-CCGG-3` sequence located between primer annealing sites is presented. Samples 1–4 (S1–S4) each represent 1 of the 4 patterns of cytosine methylation possible at a 5`-CCGG-3` sequence. In S1, neither cytosine is methylated. Thus, both MspI and HpaII will cleave the CCGG sequence and no bands will be seen in either the MspI or HpaII digest lanes. In S2, the internal cytosine is methylated so that HpaII cannot cleave, but MspI can (MspI cleaves regardless of the methylation status of the internal cytosine). In S3, the external cytosine is methylated so that MspI cannot cleave and HpaII cleaves at a much reduced rate, resulting in an intense band in the MspI digest lane, and a faint band in the HpaII digest lane. In S4, both cytosines are methylated and neither enzyme is able to cleave; therefore, bands are seen in both the MspI and HpaII digest lanes.

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphoimages of 33P-PCR products from PB-treated and untreated samples from C57BL/6, C3H/He, and B6C3F1 using the single arbitrary primer are represented in Figures 2a, 2b, and 2cGo, respectively. These results are tabularized in Table 1Go. In the tumor-resistant C57BL/6 strain (Fig. 2aGo), the banding pattern of untreated and PB-treated samples is similar. However, in each phosphoimage shown there are 2 bands seen in the PB-treated RsaI/MspI-digested samples, and in 1 phosphoimage there is 1 band seen in the PB-treated RsaI/HpaII-digested samples that are seen less distinctly in the untreated samples. This is indicative of a small amount of PB-induced hypermethylation at the external and internal cytosine of 5`-CCGG-3` sites. In the tumor-prone C3H/He strain (Fig. 2bGo), the banding pattern in the PB-treated samples is more markedly different as compared to the untreated samples. There were 5–9 bands seen in the RsaI/MspI and RsaI/HpaII lanes of PB-treated samples not seen in the untreated samples. These data are indicative of hypermethylation at numerous external and internal cytosines in 5`-CCGG-3` sites in the PB-treated C3H/He mice. The banding pattern of the B6C3F1 phosphoimage (Fig. 2cGo) was more similar to C3H/He than C57BL/6; a moderate amount of hypermethylation was seen at the external cytosine site of PB-treated samples.



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FIG. 2. Methylation status of GC-rich regions of hepatic DNA in C57BL/6 (a), C3H/He (b), and B6C3F1 (c) mice was assessed by 33P-PCR using a single arbitrary primer. RsaI/MspI and RsaI/HpaII digests are presented in lanes indicated by and M and H, respectively. Numbers underneath the bars on the top of the gel indicate individual animals. Animals 1–3 were untreated, and animals 4–6 or 7 were PB-treated. Thus, data shown is representative of 3 untreated and 3–4 treated animals. Analysis was repeated for 2 controls and 1–2 treated animals from each group to test for reproducibility. Two digests were performed per DNA sample. In rows marked A, the M lanes of treated animals are more prominent than in the controls; in rows marked B, the H lanes of treated animals are more prominent than in the controls; in rows marked C, the M lanes of treated animals are less prominent than in the controls. R indicates a reference row of bands that are reasonably constant and highlighted to illustrate loading differences. Sections of images above and below the thick black line approximately at the middle of each image are of the same gel; however, the exposure times were different in order to better visualize the separated PCR products. The lower portion represents a longer, 7–9 day exposure while the upper portion represents a shorter, 2–5 day exposure. The vertical heavy black lines alongside phosphoimages in (a) and (b) indicate regions subjected to image analysis and represented graphically in Figure 4Go.

 

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TABLE 1 Summary of Phosphoimage Data from PB-Treated and Untreated PCR Samples Using the Single Arbitrary Primer
 
Phosphoimages of 33P-PCR products from PB-treated and untreated samples from C57BL/6, C3H/He, and B6C3F1 using the arbitrary primer pair are represented in Figures 3a, 3b, and 3cGo, respectively. These results are presented in Table 2Go. In each group of PB-treated versus control mice, fewer differences were observed using the arbitrary pair of primers as compared to the data derived from the single arbitrary primer. This is likely to be due to the difference in PCR products produced by using the single primer vs. the primer pair. Unlike the primer pair, the single primer binds to a 5`-CCGG-3` site, so the status of 5`-CCGG-3` methylation may be analyzed at and between sites of primer annealing. Using the primer pair, a phosphoimage of C57BL/6 samples (Fig. 3aGo) shows 2 bands not seen in the RsaI/HpaII PB-treated lanes that were seen in the untreated lanes, indicative of a low level of hypomethylation at internal cytosine sites. There was also a very small amount of hypermethylation at the internal and external cytosine sites seen on one of the C57BL/6 phosphoimages. A small and moderate amount of hypermethylation was seen in the internal cytosines of C3H/He (Fig. 3bGo) and B6C3F1 (Fig. 3cGo), respectively. A small amount of hypermethylation at the external cytosine was seen in both B6C3F1 and C3H/He. Thus, using both the single arbitrary primer and the arbitrary primer pair, a greater amount of hypermethylation was seen in the PB-treated animals of the tumor-sensitive C3H/He and B6C3F1 mice as compared to what was seen in the C57BL/6 strain.



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FIG. 3. Methylation status of GC-rich regions of hepatic DNA in C57BL/6 (a), C3H/He (b), and B6C3F1 (c) mice was assessed by 33P-PCR using an arbitrary primer pair. RsaI/MspI and RsaI/HpaII digests are presented in lanes indicated by and M and H, respectively. Numbers underneath the bars on the top of the gel indicate individual animals. Animals 1–3 were untreated, and animals 4–6 or 7 were PB-treated. Thus, data shown in representative of 3 untreated and 3–4 treated animals. Analysis was repeated for 2 controls and 1–2 treated animals from each group to test for reproducibility. Two digests were performed per DNA sample. In rows marked A, the M lanes of treated animals are more prominent than in the controls; in rows marked B, the H lanes of treated animals are more prominent than in the controls; in rows marked C, the M lanes of treated animals are less prominent than in the controls; and in rows marked D, the H lanes of treated animals are less prominent than in the controls. R indicates a reference row of bands that are reasonably constant and highlighted to illustrate loading differences. Sections of images above and below the thick black line approximately at the middle of each image are of the same gel; however, the exposure times were different in order to better visualize the separated PCR products. The lower portion represents a longer, 7–9 day exposure while the upper portion represents a shorter, 2–5 day exposure.

 

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TABLE 2 Summary of Phosphoimage Data from PB-Treated and Untreated PCR Samples Using the Arbitrary Primer Pair
 
In order to correct for differences in the overall lane intensities, the percent intensity of radioactive signal adjusted for the overall lane intensity was ascertained (Figs. 4 and 5GoGo). In agreement with the data presented in Figure 2Go, these data indicate that there is a greater amount of PB-induced hypermethylation in the tumor-sensitive C3H/He strain as compared to the tumor-resistant C57BL/6 strain.



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FIG. 4. Analysis of regions of the C57BL/6 phosphoimage presented in Figure 2aGo. This graph illustrates the intensity of the 33P signal for points down the length (relative front) of each HpaII lane in the images after subtracting the lane backgrounds and thus correcting for differences in overall lane intensities. The pixel intensity is plotted against the relative front of each lane. Increased methylation is reflected as a greater pixel intensity.

 


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FIG. 5. Analysis of regions of the C3H/He phosphoimage presented in Figure 2bGo. This graph illustrates the intensity of the 33P signal for points down the length (relative front) of each HpaII lane in the images after subtracting the lane backgrounds and thus correcting for differences in overall lane intensities. Peaks corresponding to the first 5 B rows from the top of the image shown in Figure 2bGo are indicated. The pixel intensity is plotted against the relative front of each lane. Increased methylation is reflected as a greater pixel intensity. This is seen more prominently in the tumor sensitive C3H/He mice than in the C57BL/6 mice (see Fig. 4Go).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An arbitrarily primed PCR approach has enabled us to examine PB-induced methylation changes at GC-rich regions of DNA, including CpG islands, in 3 groups of mice varying in susceptibility to liver tumorigenesis. Using the same or similar arbitrary primers, this procedure has allowed for the identification of novel CpG islands. Gonzalgo et al. (1997) amplified a PCR product that was shown to contain a novel CpG island often methylated in bladder and colon tumors and, Kohno et al. (1998) identified CpG islands hypermethylated in human lung cancer. Using both the single arbitrary primer and the primer pair, we have discerned an overall trend toward PB-induced hypermethylation of GC-rich regions of mouse liver DNA that is more pronounced in the relatively tumor-susceptible C3H/He and B6C3F1 mice compared to the relatively resistant C57BL/6.

Highly methylated sequences in promoter regions inhibit transcription through methylated DNA-binding proteins that interfere with the binding of transcription factors to their cognate cis elements. Two such proteins, MeCP1 and MeCP2, have been found to bind preferentially to methylated DNA and repress transcription in vivo and in vitro (Bird and Wolffe, 1999Go). Furthermore, MeCP2 has been found to co-immunoprecipitate with Sin3A, a protein that interacts with histone deacetlyase (Bird and Wolffe, 1999Go). Therefore, methylated regions may be more apt to become deacetlylated, and the associated regions become more tightly wrapped around the histones, making transcription less probable. Hypermethylation at GC-rich sequences in promoter regions has been shown to be linked to the silencing of several known tumor suppressor genes, including p16 (Esteller et al., 1999Go), p14 (Esteller et al., 2001bGo), and O6-methylguanine-DNA methyltransferase (Patel et al., 2000Go), in a variety of cancers.

Previous studies performed in our laboratory examined global, average methylation status in B6C3F1 and C57BL/6 mice and demonstrated a greater amount of global hypomethylation in hepatic DNA of PB-treated animals as compared with the C57BL/6 strain (Counts et al., 1996Go). However, a limitation of this earlier study is the fact that the methodology employed did not permit us to discern treatment-induced increased methylation in particular regions of DNA. The arbitrarily primed PCR procedure used in the current investigation is advantageous because it extends the analysis of methylation change by allowing for the detection of specific methylation alterations in GC-rich regions of the genome. This analysis aids in understanding the multitude of effects of tumor promoters on methylation.

A unique feature of this study is that through the use of the methylation-sensitive enzymes MspI and HpaII we were able to detect hypermethylation occurring at both the internal and external cytosines of the 5`-CCGG-3` site. While most methylation reported to occur in mammals is located within the symmetrical dinucleotide CpG, CpNpG, methylation has been shown to occur in mammalian cells (Clark et al., 1995Go, 1997Go; Ray et al., 1994Go; Stirzaker et al., 1997Go). Using the single arbitrary primer, a greater or equivalent amount of PB-induced meCpCpG compared to meCpG was found in B6C3F1 and C3H/He. In the C3H/He strain using the arbitrary primer pair, there was no distinct difference detected in the amount of methylation at CpG and CpCpG sites. Such a difference may be more difficult to detect with the primer pair because there was less overall variation between band patterns of treated versus control samples.

It is possible that mechanisms, in particular the methyltransferases, responsible for maintenance of methylation at CpG and non-CpG sites vary. In mammalian embryonic stem cells, the level of de novo methyltransferase Dnmt3a has been correlated with the presence of non-CpG methylation (Ramsahoye et al., 2000Go). When treated with certain tumor promoters, specific methylases may be more or less affected than others leading to differences in the level of methylation change at CpG and non-CpG sites.

A tumor-promoting dose of PB has been shown to lead to an increase in liver cell proliferation 1–2 weeks after administration as well as a decrease in the levels of S-adenosyl methionine (SAM), the cofactor for methylation reactions (Shivapurkar and Poirier, 1982Go). These effects vary between C57BL/6 and B6C3F1. While the increase in liver cell proliferation is more marked in C57BL/6, the decrease in global methylation is more prominent in B6C3F1. Hepatic DNA from B6C3F1 mice also exhibits a higher level of global hypomethylation following a choline/methionine deficient diet (Counts et al., 1996Go).

Genetic differences between strains of mice are likely to contribute to these variations in the ability to maintain patterns of DNA methylation. The lifetime rate of spontaneous tumor formation is less than 5% in C57BL/6 mice, but up to 80% in C3H/He mice (Buchmann et al., 1991Go; Grasso and Hardy, 1975Go). C3H/He mice are 20–50x more susceptible than C57BL/6 mice to induction of cancer by the carcinogens N-diethylnitrosamine (Drinkwater and Ginsler, 1986Go) and N-ethyl-N-nitrosourea (Hanigan et al., 1988Go). It has been proposed that multiple susceptibility loci within an Hcs (hepatocarcinogen sensitivity site) account for approximately 85% of this difference in sensitivity (Drinkwater and Ginsler, 1986Go). Furthermore, mutational activation of Ha-ras is more often seen in C3H/He and B6C3F1 tumors than in C57BL/6 (Buchmann et al., 1991Go) and strain differences in the methylation status of this gene may play a role in the expression of this oncogene (Counts and Goodman, 1994Go).

Several factors are involved in maintaining methylation, including the availability of methyl groups, methylation cofactors including SAM, and the utilization of methyl groups by methyltransferases and demethylases. Dnmt 1 is believed to act primarily as a maintenance methylase, although it also may act as a de novo methylase (Bestor et al., 1988Go) while Dnmt 3a/3b is believed to act as a de novo methylase (Okano et al., 1999Go). A genetic variation within the multiple susceptibility loci that affects any part of any one of these factors could have a large effect on the capacity to maintain normal patterns of methylation. For instance, a strain difference in the activity of a particular methyltransferase could potentially lead to the observed changes in global and regional methylation, both of which are more markedly altered in the tumor-sensitive C3H/He and B6C3F1 mice than in the tumor-resistant C57BL/6. It is possible that Dnmt1, Dnmt3, and perhaps other methyltransferases compete for and/or have some specificity for methylation at particular regions of DNA. If a predominantly globally acting methyltransferase in C3H/He and B6C3F1 was more sensitive to dietary and PB-challenge than C57BL/6, compensatory expression of a methyltransferase acting preferentially at CpG islands may explain why we see PB induced global methylation and CpG island hypermethylation predominantly in these mice.

The results reported here in combination with previous studies on global levels of methylation (Counts et al., 1996Go) indicate that specific hypermethylation in GC-rich regions of DNA in response to a tumor-promoting dose of PB occurs concurrently with a decrease in global levels of methylation. Both hyper- and hypomethylation contribute significantly to carcinogenesis and, importantly, the simultaneous occurrence of these events is not mutually exclusive (Counts and Goodman, 1995aGo,bGo). Indeed, altered methylation may be viewed as a secondary mechanism underlying carcinogenesis (Goodman and Watson, 2002Go). Furthermore, the capacity of PB to affect DNA methylation is greater in the tumor sensitive C3H/He and B6C3F1 mice as compared to the relatively resistant C57BL/6 strain. Taken together, these sets of data support the hypothesis that susceptibility to carcinogenesis may be related inversely to the capacity to maintain normal methylation patterns.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (517) 353-8915. E-mail: goodman3{at}msu.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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