, 2006b). Cloning and annotation of these unique RAMs revealed changes in methylation which occurred within genes that are known to play important roles in tumorigenesis (e.g., research use angiogenesis, invasion, metastasis, and epithelial-mesenchymal cell transition), plus genes which had not previously been linked to cancer, thus providing insight regarding specific genes which may play a role in PB-induced tumorigenesis due to altered DNA methylation (Phillips and Goodman, 2008). In an analogous fashion to Bachman et al. (2006b), Phillips et al. (2007) compared DNA methylation patterns in liver tumor�Csusceptible C3H/He CAR WT mice and resistant CAR KO mice. Unique RAMs in the livers of CAR WT mice initiated with DEN and treated with PB for 23 (precancerous tissue) or 32 (tumor tissue) weeks, compared with CAR KO mice initiated with DEN and treated with PB for 23 weeks, were identified.

Methylation changes also occurred in the CAR KO, PB-treated mice, suggesting, analogous to gene expression changes observed in Ueda et al. (2002), that DNA methylation changes are both CAR dependent and independent. We hypothesize that a subset of the unique RAMs in the precancerous and tumor tissue are important for facilitating tumorigenesis. In this study, unique RAMs detected in the precancerous and tumor tissue (Phillips et al., 2007) were cloned and annotated to discern the particular genes involved and how they might contribute to tumorigenesis, for example, common cellular targets of the genes of interest were identified in order to picture how they might interact to affect critical signaling pathways, leading to key alterations in phenotype.

Although Phillips and Goodman (2008) focused upon genes involved in PB-induced tumor formation at very early treatment times (i.e., 2 and 4 weeks), the current study elucidated genes involved at later times, when foci (23 weeks, PB-treated CAR WT) and tumors (32 weeks, PB-treated CAR WT) are apparent. Taken together, these two studies (the aforementioned B6C3F1-C57BL/6 study, and the current CAR study) lead to the identification of genes whose methylation statuses changed uniquely in liver tumor�Csusceptible mice (B6C3F1 and CAR WT), as compared with their resistant counterparts (C57BL/6 and CAR KO, respectively), within a continuum of PB-induced tumorigenesis.

MATERIALS AND METHODS Animals, Treatments, and Tissue Samples The DNA employed for these studies was isolated from the same liver samples used by Phillips et al. (2007), and these samples were provided by Yamamoto et al. (2004). CAR WT or CAR KO mice, on a C3H/He background (which is highly susceptible to liver tumorigenesis (Buchmann et al., 1991), were injected with a single Dacomitinib intraperitoneal dose of DEN, 90 mg/kg, at 5 weeks of age and then administered drinking water (control) or 0.