Fig. 2

Age-dependent p16 epimutation cooperates with Apc mutation to promote colon cancer. A Accelerated methylation of p16^cis alleles compared with WT alleles in intestinal tissues during aging as determined by bisulfite pyrosequencing. B Bisulfite PCR, cloning, and sequencing show a clonal expansion pattern associated with p16 promoter hypermethylation in aged intestines. Each row represents a single sequenced molecule. Black and white circles represent methylated and unmethylated CpGs, respectively. C The addition of p16 epimutation in Apc-mutant mice shortened survival. Survival was compared using the Kaplan–Meier method in Apc^Min/+ mice (10 males and 16 females) and Apc^Min/+; p16^cis/cis mice (15 males and 14 females). D At 15 wk of age, Apc^Min/+; p16^cis/cis mice developed more tumors in the distal small intestines compared to Apc^Min/+ and Apc^Min/+; p16^cis/+ mice. E Tumor number vs. size in the distal small intestines. F More colon tumors were found in the Apc^Min/+; p16^cis/cis mice at 15 wk of age compared to the other two groups. For figures (D–F), data are mean ± SEM with individual values. P values were determined by a two-tailed Student’s t-test. G H&E staining of the colon tumor sections revealed histologic features of malignant transformation in Apc^Min/+; p16^cis/cis mice. Yellow lines indicate tubular adenomas with extensive high-grade dysplasia, and arrows indicate focally invasive adenocarcinoma. Scale bars: 100 μm