Identification of genes that may play critical roles in phenobarbital (PB)-induced liver tumorigenesis due to altered DNA methylation.

Aberrant DNA methylation plays important roles in tumorigenesis, and the nongenotoxic rodent tumor promoter phenobarbital (PB) alters methylation patterns to a greater extent in liver tumor susceptible as compared to resistant mice (Watson and Goodman, 2002). Unique hepatic regions of altered DNA methylation (RAMs) were identified in sensitive B6C3F1, as compared to resistant C57BL/6, mice at 2 or 4 weeks of PB treatment using a novel approach involving methylation-sensitive restriction digestion, arbitrarily primed PCR, and capillary electrophoresis (Bachman et al., 2006b). PCR products representing 90 of 170 (53%) total unique B6C3F1 RAMs at 2 or 4 weeks were cloned and subjected to BLAST-like alignment tool searches that resulted in 51 gene matches; some of these have documented oncogenic or tumor suppressor roles. Importantly, uniquely hypomethylated genes play roles in angiogenesis (e.g., chymase 1, tyrosine kinase nonreceptor 2, and possibly ephrin B2 and triple functional domain, PTPRF interacting) and invasion and metastasis, including those involved in the epithelial-mesenchymal transition (transcription factor 4, transforming growth factor beta receptor II, and ral guanine nucleotide dissociation stimulator). Common cellular targets and regulators of the genes representing unique B6C3F1 RAMs were uncovered, indicating that they might act in concert to more efficiently promote tumorigenesis. Genes not previously associated with mouse liver tumorigenesis exhibited altered methylation at these very early times following PB treatment. We hypothesize that at least some of the unique PB-induced B6C3F1 RAMs represent key events facilitating transformation, which is consistent with a causative role of altered DNA methylation during early stages of tumorigenesis.