Fig. 2.

Molecular analyses of PDAC cell lines and primary tumor specimens from mice with p53 and p16 mutant animals. (A) PCR reactions to detect the p53-WT (+) and p53^lox alleles (Upper) and p53-null (−) allele (Lower) in normal tissue from p53^lox/lox (lane 1) and p53^lox/+ (lane 7) mice and tumor cell lines (lanes 2–6). All tumor cell lines show only the p53-null allele. Germ-line p16^Ink4a status is indicated at the top. (B) Western blot for p16^Ink4a and p19^Arf expression in cell lines derived from p53^lox/lox mice with various p16^Ink4a genotypes. The negative and positive controls are in lanes 9 and 10, respectively. α-Tubulin is shown as a loading control. (C) PCR analysis of the p53⁺ and p53^lox alleles (Upper) and the p53⁻ (/) allele (Lower) demonstrates loss of the p53⁺ allele in all tumors from p53^lox/+ p16^+/− mice (lanes 2–6). Lane 1 shows the WT control specimen. (D) Western blot analysis shows low or absent p16^Ink4a expression in five of six tumor cell lines from p53^lox/+ p16^+/− mice (Right, lanes 1–6), whereas all retain p19^Arf expression. Negative controls are in lanes 7 and 12. p16^Ink4a expression in PDAC cell lines from p53^lox/lox p16^+/+ (lane 8) and p53^lox/lox p16^+/− (lane 10) mice is shown as a reference (Right). (E) PCR analysis of the p16^Ink4a− and p16^Ink4a+ alleles demonstrates LOH in one of six samples (lane 5). Normal tissue specimens are shown as controls for the p16^Ink4a+/+, p16^Ink4a−/−, and p16^Ink4a+/+ alleles (lanes 1–3). (F) Methylation-specific PCR assay to detect methylated CpG islands in the p16^Ink4a promoter region reveals hypermethylation in three tumor lines (lanes 1, 4, and 5). Upper and Lower show the methylated (M) and unmethylated (U) alleles, respectively. Negative and positive controls are in lanes 7 and 8.