Department of Pharmacology and Toxicology, Michigan State University, B440 Life Sciences Building, East Lansing, Michigan 48824
Received January 7, 2002; accepted March 6, 2002
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ABSTRACT |
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Key Words: B6C3F1; carcinogenesis; C3H/He; C57BL/6; CpG islands; DNA methylation; GC-rich regions; liver; non-CpG methylation; phenobarbital; susceptibility; tumorigenesis.
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INTRODUCTION |
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In general, the density of methylation is related inversely to gene expression (Laird and Jaenisch, 1994). 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., 1998
; Nan et al., 1998
). 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, 2000
). 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., 1999
). Similarly, methylation of a differentially methylated nonpromoter region is necessary for paternal expression of Igf2 (Bell and Felsenfeld, 2000
). 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, 2000). More specifically, hypermethylation in promoter regions of tumor suppressors, associated with silencing of these genes, is a common finding in cancer (Lin et al., 2001
). 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., 2001a
; Patel et al., 2000
). Similarly, loss of pRb expression in pituitary adenocarcinomas has been shown to be associated with methylation of RB1 promoter regions (Simpson et al., 2000
). 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., 1994
). Furthermore, hypomethylation could lead to a decrease in genomic integrity by reducing the methylation-mediated silencing of foreign genomic elements (Laird, 1997
).
Consistent with the observation of global hypomethylation in tumor samples and precancerous lesions as compared to normal tissue (Gama-Sosa et al., 1983), 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., 1996
).
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., 1997). 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.
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MATERIALS AND METHODS |
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DNA isolation and restriction digests.
DNA was extracted by a phenol/chloroform procedure (Strauss, 1990). 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, 1977
). 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., 1997). 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 25 days, followed by a longer exposure of 59 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 751500 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 1. We considered a control versus treatment group difference of 13, 46, 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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RESULTS |
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DISCUSSION |
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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, 1999). Furthermore, MeCP2 has been found to co-immunoprecipitate with Sin3A, a protein that interacts with histone deacetlyase (Bird and Wolffe, 1999
). 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., 1999
), p14 (Esteller et al., 2001b
), and O6-methylguanine-DNA methyltransferase (Patel et al., 2000
), 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., 1996). 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., 1995, 1997
; Ray et al., 1994
; Stirzaker et al., 1997
). 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., 2000). 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 12 weeks after administration as well as a decrease in the levels of S-adenosyl methionine (SAM), the cofactor for methylation reactions (Shivapurkar and Poirier, 1982). 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., 1996
).
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., 1991; Grasso and Hardy, 1975
). C3H/He mice are 2050x more susceptible than C57BL/6 mice to induction of cancer by the carcinogens N-diethylnitrosamine (Drinkwater and Ginsler, 1986
) and N-ethyl-N-nitrosourea (Hanigan et al., 1988
). 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, 1986
). Furthermore, mutational activation of Ha-ras is more often seen in C3H/He and B6C3F1 tumors than in C57BL/6 (Buchmann et al., 1991
) and strain differences in the methylation status of this gene may play a role in the expression of this oncogene (Counts and Goodman, 1994
).
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., 1988) while Dnmt 3a/3b is believed to act as a de novo methylase (Okano et al., 1999
). 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., 1996) 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, 1995a
,b
). Indeed, altered methylation may be viewed as a secondary mechanism underlying carcinogenesis (Goodman and Watson, 2002
). 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.
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NOTES |
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