Altered methylation in gene-specific and GC-Rich regions of DNA is progressive and nonrandom during promotion of skin tumorigenesis

Altered DNA methylation, an epigenetic mechanism, likely contributes to tumorigenesis, with an inverse relationship existing between methylation in a promoter region and transcription. Using the SENCAR two-stage mouse skin tumorigenesis model, altered methylation was characterized in precancerous tissue and in tumor tissue. Mouse skin was initiated with 7,12-dimethylbenz[a]anthracene and promoted three times a week with 3, 9, 18, or 27 mg cigarette smoke condensate (CSC) for 4, 8, or 29 weeks; tumors were collected at 29 weeks. In addition, reversibility of changes in methylation was assessed following cessation of the promoting stimulus. DNA was isolated, and GC-rich methylation was assessed quantitatively via methylation-sensitive restriction digestion, arbitrarily primed PCR, and electrophoretic separation of PCR products. Analysis focused on regions of altered methylation (RAMs), which persisted from 4 to 8 weeks and from 8 weeks to tumor tissue. Persistent RAMs (i.e., seen in precancerous tissue and carried forward to tumors) are likely to play a key role in tumorigenesis. Twenty-two CpG sites in the upstream region of the Ha-ras promoter were unmethylated in control skin, 27 mg CSC, and tumor tissue. At two CpG sites closer to the transcriptional start site the incidence of hypomethylation increased with the dose of CSC. Hypomethylation was detected in all tumor samples. Expression of Ha-ras increased with 18 and 27 mg CSC promotion and more so in tumor tissue. These data support our hypothesis that tumor promotion involves instability of the epigenome, providing an environment where changes in the methylation status of specific regions of the genome accumulate progressively and contribute to the clonal expansion of initiated cells that leads to tumor formation.