Casanovas O, Hicklin DJ, Bergers G, Hanahan D. c-is estimated to be mutated in ~9% of NSCLCs, 25% of VU 0240551 which express the oncogene encoding the BRAFV600E oncoprotein kinase (4C6). However, like many cancers, mutational activation of proto-oncogenes such as or is generally accompanied by silencing VU 0240551 of tumor suppressor genes (TSGs) such as or that cooperatively serve to promote the step-wise malignant transformation of normal lung epithelial cells to malignant lung cancer cells (5,6). To model BRAFV600E-induced lung tumorigenesis, we previously generated mice engineered to carry a Cre-activated allele that allows conversion of normal BRAF to BRAFV600E following exposure of cells to viruses encoding Cre recombinase (7,8). Expression of BRAFV600E in the distal lung epithelium results in development of benign lung tumors that fail to progress to lung cancer due to the onset of a senescence-like proliferative arrest (8, 9). Importantly, when TSGs or proto-oncogenes (mice to model BRAFV600E-induced tumorigenesis (8C10), we are constrained by our inability to temporally dissociate genetic events that contribute to cancer progression. Somatic recombination of conditional alleles by Cre recombinase is such that oncogene activation and TSG silencing occur simultaneously C a situation that rarely occurs in humans (11). We therefore wanted to generate a new GEM model of lung cancer in which expression of BRAFV600E could be temporally dissociated from cooperating genetic events that contribute to malignant progression. To do so, we generated mice carrying a Flp-activated allele of (function prior to acquiring oncogenic mutations such as that observed in Li-Fraumeni patients (12,13), we VU 0240551 next modeled this phenomenon by inducing BRAFV600E expression after TP53 silencing. This order of events appeared to modestly enhance the aggressiveness of the disease. To explore the consequences of TP53 silencing in lung cancer cells, we generated BRAFV600E/TP53Null lung cancer cell lines in which we could restore TP53 activity. Restoration of TP53 activity did not result in VU 0240551 senescence or apoptosis, but in a reversible G1 cell cycle arrest that was independent of p19ARF expression. These results highlight the growing sophistication of GEM models of human cancer and demonstrate the importance of TP53 signaling in restricting malignant progression of BRAFV600E-induced benign lung tumors. MATERIALS AND METHODS Strains of mice and Adenoviral Infections The following strains of mice have been previously described: aka (8)), (((15)), (((aka mice To generate mice, a targeting vector was made by exchanging the two sites in the original targeting vector with sites using VU 0240551 standard cloning techniques (Fig. 1A) (8). By homologous recombination, we generated ES cells and confirmed correct targeting of by Southern blot analysis of ES cell genomic DNA as described previously (Fig. 1BCD) (8). One of these ES clones was injected into mouse blastocysts, which in turn gave rise to a chimeric mouse that transmitted the allele through the germ-line. The resulting progeny were used for further experimental studies. Open in a separate window Figure 1 Generation of miceA. Schematic representation of the targeting construct used to generate the mouse through homologous recombination into the endogenous genomic locus of 129SvEv derived ES cells. B. Schematic of the structure of the allele that expresses normal BRAF and NEO prior to Flp-mediated recombination. P1 and P2 indicate the location of LIF Southern blotting probes used to confirm appropriate targeting of the gene. C. Schematic of the structure of the allele following Flp-mediated recombination that now expresses mutationally activated BRAFV600E. D. Southern blot analysis to identify ES clones with the targeting construct. E. H&E stained sections of (left) and (right) 10 weeks p.i. with Ad-Flp or Ad-Cre respectively with average tumor burden indicated in the bar graph. F. Lung sections of tumor-bearing or mice stained with H&E, Ki67, anti-SPC/CC10 or AQP5 using either IHC and IF methods as indicated. G. Immunoblot analysis of lung tumor lysates from or mice 10 weeks.