* Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824, and
R. J. Reynolds Tobacco Company, Research and Development, Winston-Salem, North Carolina 27102
Received April 17, 2003; accepted June 28, 2003
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ABSTRACT |
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Key Words: DNA methylation; tumorigenesis; promotion; SENCAR mouse; skin tumorigenesis; epigenetics.
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INTRODUCTION |
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Changes required for the basic cancer phenotype include evasion of apoptosis, self-sufficiency in growth signals, insensitivity to antigrowth signals, tissue invasion, and metastasis, limitless replicative potential, and sustained angiogenesis (Hanahan and Weinberg, 2000). While these events might occur via mutation, epigenetic events can play a fundamental role in carcinogenesis (Watson and Goodman, 2002b
). Epigenetic regulation of gene expression occurs through heritable transcriptional modulation superimposed on the primary DNA sequence. Thus, epigenetic mechanisms such as DNA methylation, i.e., the 5-methylcytosine content of DNA, have the capacity to change transcriptional levels without changing the sequence (Holliday, 1994
). Genes commonly found to have altered transcriptional levels in cancer, such as the often underexpressed tumor suppressor p53 and the often overexpressed oncogene ras, can be altered by mutation or epigenetic mechanisms (Hosaka et al., 2002
). Importantly, a mutated oncogene needs to be expressed in order to contribute to carcinogenesis (Hahn et al., 1999
), and the expression level might be governed by epigenetic mechanisms.
Methylation facilitates a remodeling of chromatin to an inactive state. Increased methylation in GC-rich promoter regions of genes is generally associated with decreased transcription and vice versa (Ballestar and Esteller, 2002). Much of the promoter-specific methylation occurs at CpG islands, 200 bp or longer stretches of DNA with a 50% or greater GC content and a higher than expected CpG content (Gardiner-Garder and Frommer, 1987
). In several types of cancers, increased methylation in the promoter regions of tumor suppressors such as p16, E-cadherin, and O6-methylguanine DNA methyltransferase (MGMT) is associated with, and believed to be the cause for, decreased expression of these genes (Esteller et al., 2001
). Both hyper- and hypomethylation may contribute to carcinogenesis via silencing of tumor suppressor genes, upregulation of oncogenes, and/or decreased genome stability (Counts and Goodman, 1995
; Goodman and Watson, 2002
). Tumors characteristically exhibit increases in methylation at GC-rich regions with a decreased overall or global methylation (Gama-Sosa et al., 1983
) that can facilitate oncogene expression. Changes in methylation precede tumor formation, indicating that these alterations might contribute to tumorigenesis (Robertson and Jones, 2000
). There has been limited research on mouse skin methylation, but a few reports indicate methylation differences between normal and tumor skin tissue (Ramsden et al., 1985
; Winter et al., 1990
).
We have examined both global and GC-specific methylation using a SENCAR mouse skin initiation/promotion model of tumorigenesis. The SENCAR mouse stock was generated in the 1960s and 1970s from selective breeding of mice sensitive to epidermal papilloma formation in response to 7,12-dimethylbenz[a]anthracene (DMBA) initiation and the croton oil (containing TPA) promoting agent (Stern and Conti, 1996). These mice are extremely sensitive to carcinogenesis, and generally respond more rapidly and uniformly to the induction of skin tumors than other available strains or stocks. Importantly, the initiation and promotion stages are clearly demarcated, thus facilitating the study of biochemical and molecular mechanisms involved in a particular stage of carcinogenesis (Slaga et al., 1996
).
SENCAR mice were initiated with a dermal application of DMBA, followed by administration of various doses of cigarette smoke condensate (CSC), a presumptive tumor promoter, for different lengths of time. We are testing the hypothesis that specific types of methylation alterations play a role during the promotion stage of carcinogenesis. Four specific aims were addressed: (1) to assess methylation status during tumorigenesis in this classic two-stage model system, (2) to ascertain whether particular methylation changes correlate to tumor formation in a sequential fashion, (3) to determine whether changes in methylation exhibit a dose-response relationship with regard to promoter treatment, and (4) to assess the potential for reversibility of altered methylation in precancerous tissue.
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MATERIALS AND METHODS |
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Global DNA methylation analysis using the SSSI methylase assay.
SssI methylase utilizes S-adenosyl methionine as a methyl group donor to methylate the 5' position of cytosine at unmethylated CpG sites in DNA. Thus, the level of global DNA methylation can be determined by the amount of tritiated methyl groups from [3H-CH3] S-adenosyl-L-methionine incorporated into DNA, since there is an inverse relationship between incorporation of radioactivity and the original degree of methylation. DNA (1 mg) was incubated with 2 µCi [3H-CH3] S-adenosyl-L-methionine (New England Nuclear, Boston, MA) and 3 units of SssI methylase (New England Biolabs, Beverly, MA) for 1 h at 30°C. Results are presented as counts per min per microgram (cpm/µg) DNA. Five replicates were performed per sample. Graphical presentation was performed using Excel®. Statistical analysis was performed with Excel using 2-tailed t-tests to compare the average cpm/µg DNA measurements between treatment groups and controls. A p value of <0.05 was considered statistically significant.
Methylation Analysis of GC-Rich Regions
Restriction digests.
For each DNA sample, three restriction digests were performed as follows: RsaI alone, RsaI and MspI, and RsaI and HpaII. RsaI is a methylation-insensitive enzyme used to cut the DNA into smaller fragments. Both MspI and HpaII are methylation-sensitive enzymes that cut between cytosine residues at 5'-CCGG-3' sites. MspI will not cut if the external cytosine is methylated, while HpaII will not cut if the internal cytosine is methylated; both will cut if the site is unmethylated (Mann and Smith, 1977). All enzymes used were from Roche (Indianapolis, IN). Restriction digests were performed with 1 µg of DNA and 5.0 units of RsaI in Roche 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 primer (5'-AACCCTCACCCTAACCCCGG-3') that arbitrarily binds within GC-rich regions of DNA (Gonzalgo et al., 1997). Reactions were composed of 5 ml of the restriction digest (containing 1 mg digested DNA), 0.4 µM each primer, 1.25 units of Taq polymerase (Gibco BRL, Rockville, MD), 1.5 mM MgCl2, 60 mM Tris, 15 mM ammonium sulfate, 1.65 mCi
-33P-dATP (New England Nuclear, Boston, MA), and glass-distilled water to volume. Samples were heated for 5 min at 94°C before addition of dNTPs in order 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 under the following conditions: 30 s at 94°C, 1 min at 40°C, 1.5 min at 72°C; then 30 cycles of 94°C for 30 s, 55°C for 15 s, and 72°C for 1 min, a time delay cycle for 5 min at 72°C, and a soak cycle at 4°C. PCR products (5 µl of each) were separated on a 6% polyacrylamide sequencing gel at 45 watts for 2
2
h. The gel was soaked for 10 min in a fixing solution with 5% acetic acid and 5% methanol, rinsed for 10 min in glass-distilled water, dried, and placed into a cassette with a storage phosphoimage screen to visualize labeled PCR products. Compared to larger DNA fragments on the upper halves of gels, smaller fragments on the lower halves of gels sometimes required a longer exposure to clearly discern bands. Thus, a short exposure of 3 days followed by a longer exposure of 8 days was often performed on a gel. Phosphoimages were analyzed using Quantity One® Bio-Rad software.
Quantification of band intensity.
Regions of the phosphoimages in which bands appear more or less prominently compared to controls were boxed and numbered. Bands within these regions were outlined and measured for pixel number and intensity using NIH image. The total pixel intensity units for each band were obtained by multiplying the pixels in the band by the mean intensity units within the outlined region. Reference rows of bands (R) with reasonable lane-to-lane consistency were chosen to represent lane-to-lane background and/or loading differences. In order to compensate for any differences in lane background levels, the ratio of band intensity for each numbered region to the band intensity within the corresponding lanes reference (R) region was determined. Ratio fold change differences between CSC-promoted and control animals were calculated by dividing the ratios of the CSC-promoted animals by the ratios of the control animals in corresponding regions.
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RESULTS |
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In order to determine whether the effect of promoter treatment requires initiation to induce increases in methylation, the GC-rich methylation patterns of non-tumor tissue collected at 9 weeks from animals initiated with DMBA and promoted with 27 mg CSC (group 9) were compared with non-tumor tissue from animals initiated with acetone and similarly promoted with CSC (group 13). Regardless of whether the skin was initiated or not, increases in methylation were detected at the external cytosine site, indicating that prior application of an initiator was not necessary for CSC to effect methylation at the 9 week time point (data not shown). Furthermore, there were no differences seen in the GC-rich methylation patterns between DMBA-initiated, acetone-promoted (9 or 15 weeks), and untreated (29 weeks) animals (data not shown).
Next, we examined GC-rich methylation in non-tumor tissue from animals treated with various doses following 6 (Fig. 4) and 9 (Fig. 5
) weeks promotion to determine the concentration of CSC necessary to induce detectable changes at each time point. The lowest dose found to cause changes in GC-rich methylation at 6 weeks was 27 mg (Fig. 4b
). At this dose, there were 2 regions in both RsaI/MspI and RsaI/HpaII lanes at which bands were more prominent in CSC-promoted animals compared to acetone-promoted controls. The corresponding increases in pixel intensity ratios of the promoted animals (Fig. 4c
) are indicative of increased methylation at both the internal and external cytosine sites. There were also two regions at which bands were seen less prominently in the RsaI/MspI lanes of the CSC-promoted animals compared to controls. The corresponding decreases in pixel intensity ratios (Fig. 4c
) are indicative of a decrease in methylation at the external cytosine site. No changes in GC-rich methylation were detected with 18 mg CSC promotion at 6 weeks (Fig. 4a
). Thus, the threshold dose needed to elicit detectable changes in GC-rich methylation at 6 weeks is between 18 and 27 mg CSC.
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In order to determine if CSC-induced changes in GC-rich methylation were reversible, we compared the effects of a 9-week treatment of 27 mg (group 9, Fig. 6a), a 9-week treatment of 27 mg followed by 6 weeks of no treatment (group 10, Fig. 6b
), and a 27-mg treatment for 15 weeks (group 11, Fig. 6c
) in non-tumor tissue. In all of these groups, an increased amount of methylation at the external cytosine was observed. More prominent increases in external cytosine methylation were observed for treatment groups 9 and 11 compared to those seen for treatment group 10, consistent with the finding that the CSC-induced changes in GC-rich methylation are reversible. Table 1
presents quantification of the phosphoimages depicted in Figure 6
, showing that the most prominent increases in pixel intensity ratios in promoted animals compared to corresponding controls are seen in animals treated with 27 mg for 9 and 15 weeks (Figs. 6a and 6c
, respectively; note in particular regions 1 and 4 in Table 1
) in comparison to animals which were promoted with 27 mg CSC for 9 weeks and sacrificed after a 6-week recovery period (Fig. 6b
; note region 3 in Table 1
). These data support the conclusion that the increases in methylation induced by CSC are reversible.
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DISCUSSION |
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Maintaining normal patterns of methylation is dependent on multiple, interdependent factors including maintenance and de novo methylation, demethylation not linked to DNA replication, the availability of methyl group sources (S-adenosyl methionine is the proximate methyl donor), cellular proliferation, and cellular differentiation (Goodman and Watson, 2002). Alteration of one or more of these factors may lead to hyper- and/or hypomethylation, both of which have been shown to contribute to carcinogenesis. Maintenance methylation following DNA replication is accomplished by Dnmt1, which acts preferentially at hemimethylated sites in DNA, while de novo methylation is primarily accomplished by Dnmt 3a and b (Okano et al., 1998
). Changes in the cellular proliferation rate challenge a cells methylation machinery to adjust the maintenance methylation rate accordingly, and cellular differentiation is controlled by changes in DNA methylation. In addition, methylation can be directed to specific regions of DNA. For example, the leukemia-promoting promyeloid leukemia retinoic acid receptor fusion protein has been shown to induce gene hypermethylation and silencing by recruiting DNA methyltransferases to target promoters (DiCroce et al., 2002
). A unique feature of our study is that through the use of HpaII and MspI, methylation of both the internal and external cytosines of the 5'-CCGG-3' site was assessed. We report that the majority of persistent methylation changes found within the GC-rich regions occur at the external C of 5'-CCGG-3' sites, indicating that CSC promotion may have a targeted effect on this particular type of methylation. While the bulk of methylation research focuses on methylation within the symmetrical CpG dinucleotides, CpNpG methylation has been detected in mammalian cells (Clark et al., 1997
; Stirzaker et al., 1997
). The specific basis for CpNpG methylation in mammalian systems is not known. However, in Arabidopsis, CpNpG-specific methylation occurs through an interaction of the DNA methyltransferase with histone 3, which first must be methylated by a specific methyltransferase (Jackson et al., 2002
).
Altered methylation of the GC-rich promoter regions of genes is a common event in carcinogenesis and is detectable prior to the appearance of a clinically evident tumor (Lehmann et al., 2002). For instance, methylation of the promoter regions of p16 and MGMT tumor suppressor genes has been detected in the sputum DNA of all patients with squamous cell carcinoma of the lung up to 3 years before clinical diagnosis (Palmisano et al., 2000
). Furthermore, methylation of tumor suppressor genes p16, MINT1 (methylated in tumor 1), MINT2, MINT31, MGMT, or hMLH1 is frequently observed in colorectal cancer (Chan et al., 2002
). Increases in promoter methylation of at least one of these genes was the only molecular abnormality identified in 16% of aberrant crypt foci, which are postulated to be the earliest precursor lesions in colorectal carcinogenesis (Chan et al., 2002
). The arbitrarily primed PCR procedure used in our study has been shown to amplify GC-rich, CpG-containing promoter regions of a variety of genes (Gonzalgo et al., 1997
; Kohno et al, 1998
).
Both hyper- and hypomethylation of promoter regions might contribute to carcinogenesis by facilitating the transcriptional silencing of suppressor genes and enhanced expression of oncogenes, respectively (Laird, 1997). Furthermore, hypomethylation of non-promoter regions may lead to a decreased stability of the genome due to an increase in the expression of transposons that are typically silenced by methylation (Robertson and Jones, 2000
). Therefore, alterations in DNA methylation may play a variety of roles in carcinogenesis (Counts and Goodman, 1995
).
The SENCAR mouse skin model allows for demarcation of the initiation and promotion stages of carcinogenesis (Slaga et al., 1996). Additionally, the rate of tumor formation in animals treated with initiator only has been shown to be virtually the same as that in untreated animals (Ewing et al., 1988
). Consistent with this observation, our studies demonstrate a clear dose-response relationship for tumor formation following promotion with CSC, while treating uninitiated animals with a high dose of promoter resulted in minimal tumor incidence, indicating that CSC does not appear to possess a significant initiating potential (Fig. 7
). Therefore, CSC appears to be acting primarily as a tumor promoter, and the SENCAR model is ideal for permitting examination of both qualitative and quantitative effects on methylation during the promotion stage. We have demonstrated that the promoter effects on GC-rich methylation exhibit a threshold. Moreover, threshold doses required for detectable GC-rich methylation decreased with increased time of promotion, indicating that the effects of the promoter were both time- and dose-dependent, and that the altered methylation observed fits well with the classic criteria for a mechanism involved in tumor promotion (Pitot and Dragan, 1991
, 1994
). The promoting effects of CSC on methylation are similar to those elicited by the classic rodent liver tumor promoter phenobarbital (PB), which also causes global hypomethylation (Counts et al., 1996
) and hypermethylation of GC-rich regions at both the external and internal cytosine sites at 5'-CCGG-3' sequences (Watson and Goodman, 2002a
). In addition, the effects of both PB and CSC are reversible, a hallmark characteristic of a tumor promoter (Pitot and Dragan, 1991
, 1994
).
Furthermore, we have found that the threshold dose required to induce detectable changes in GC-rich methylation (18 mg, Fig. 5) at 9 weeks is the same threshold dose required to elicit a dramatic increase in tumor incidence at 29 weeks (Fig. 7
). This suggests that methylation changes at early times might be predictive of future tumorigenesis. Indications that methylation changes might serve as biomarkers of carcinogenesis have become increasingly prevalent. For instance, it has been reported that aberrant methylation of p16 is an early event in lung cancer and a potential biomarker for early diagnosis (Belinsky et al., 1998
). Here, the GC-rich alterations detected prior to global decreases in methylation might be indicative of methylation-mediated silencing of particular tumor suppressor genes, followed by facilitation of expression of oncogenes and transposable elements. This model supports a causal role for altered methylation in skin tumorigenesis. CSC acts as a classic promoter, inducing methylation changes in a progressive, threshold-exhibiting, and reversible manner, as expected for a mechanism underlying tumor promotion. It is important to stress the fact that methylation change(s) per se, particularly at early times following chemical treatment, do not indicate that tumor formation is inevitable, since these changes are potentially reversible.
Carcinogenesis involves a progressive clonal selection/expansion of cells that are increasingly abnormal, both genetically and phenotypically. The specific sequence by which key heritable alterations to the genome occur may be an important determinant of carcinogenesis. However, it appears likely that the individual crucial alterations to critical genes stem from a stochastic process, and one can expect this to be enhanced under conditions where control of DNA methylation is decreased. Indeed, whether a particular modification predominates, e.g., hypo- versus hypermethylation and/or alterations in global and/or GC-rich regions, at a certain stage of tumor development can depend upon the species, target organ, and chemicals involved (Counts and Goodman, 1995). The current characterization of stepwise, progressive, promoter-induced alterations in methylation in the SENCAR two-stage mouse skin tumorigenesis model provides further support for the multiple roles that aberrant methylation may play in this process. Multiple changes in methylation are observed during CSC tumor promotion; increased methylation of GC-rich regions precedes global decreased methylation. Hence, progressive alterations in global and GC-rich methylation appear to be mechanistically important in tumor promotion.
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ACKNOWLEDGMENTS |
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NOTES |
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