Figure 5.
Molecular analysis of murine pancreatic adenocarcinomas. (A) Ras activation assay. Lysates from wild-type pancreas (lanes 1,2), Pdx1-Cre; LSL-KrasG12D pancreas (lanes 3,4), and the murine pancreatic adenocarcinomas (lanes 5,6) affinity precipitated with Raf RBD agarose (Upstate) and then subjected to immunoblot analysis with anti-Ras antibodies. (B) PCR analysis of the Ink4a/Arf locus in murine pancreatic adenocarcinoma cell lines. Multiplex PCR was performed on DNA from the pancreatic cancer cell lines (lanes 3-16) with primers that amplify the Ink4a/Arf+ (lower band), Ink4a/Arf lox (middle band), and Ink4a/Arf- (upper band) alleles. DNA from Ink4a/Arf+/+ (+/+, lanes 1,18) and Ink4a/Arflox/lox (L/L, lanes 2,17) mice served as controls. All cell lines show only the Ink4a/Arf- allele. (C) Immunoblot analysis of the tumor lysates. Membranes were immunoblotted for p16Ink4a, p19Arf, Smad4, and α-tubulin (as a loading control). Lysates from primary mouse embryonic fibroblasts (MEF, lane 1) served as a positive control. (D) Immunoblot analysis of p53 expression. Primary MEFs (lane 1) and p53-/- MEFs (lane 2), were positive and negative controls, respectively. (E) Induction of p53 and p21 in pancreatic adenocarcinoma cells by ionizing irradiation. Mouse pancreatic cancer cell lines were either untreated (-) or gamma irradiated (+; lanes 1-8). Lysates were immunoblotted for p53, p21, and α-tubulin. MEFs with a mutant p53 allele (p53*) were a control for p53 overexpression. Note that the tumors show modest expression of p53 compared with that of cells with mutant stabilized p53 and that ionizing radiation can effectively induce p53 and p21 in these tumor cells. (F) Amplification of the Kras gene and elevated Kras protein expression in a subset of pancreatic adenocarcinomas. The upper panel shows the relative Kras gene copy number as measured by quantitative real-time PCR. Wild-type specimens have a ratio of 1.0; -indicates not done. The middle and lower panels show Western blot analysis of the corresponding Kras levels and the tubulin (tub) loading control, respectively. Lane 1 is a control MEF specimen. Lanes 2-15 are tumor cell line specimens. Note that lanes 10 and 14 show both high-level Kras gene amplification and protein overexpression. (G) The mutant Kras allele is amplified in tumors showing increased Kras gene copy number. RT-PCR/RFLP analysis was performed on pancreatic adenocarcinoma cell line RNA to evaluate the whether the wild-type and KrasG12D alleles are expressed based on the KrasG12D-specific HindIII site. PCR-amplified cDNA was untreated (-) or digested with HindIII (+). Lanes 1-12 are tumor cell lines. Lanes 13 and 14 are control testes cDNA. All tumors express both alleles. Note that tumors 58 and 65 (lanes 2,4)—corresponding to lanes 10 and 14 in F—show an increased relative ratio of the lower KrasG12D allele, consistent with amplification and overexpression of this mutant allele.