High metastatic potential in mice inheriting a targeted p53 missense mutation

Abstract

To understand the relevance of p53 missense mutations in vivo, we generated a mouse containing an arg-to-his substitution at p53 amino acid 172, which corresponds to the R175H hot-spot mutation in human tumors by homologous recombination. Inadvertently, this mouse contains the additional deletion of a G nucleotide at a splice junction that attenuates levels of mutant p53 to near wild-type levels. Mice heterozygous for the mutant allele differed from p53(+/-) mice in tumor spectrum, with a significant increase in the number of carcinomas and a slight decrease in the number of lymphomas. More importantly, the osteosarcomas and carcinomas that developed in these mutant mice frequently metastasized (69% and 40%, respectively). In contrast, metastasis is rare in osteosarcomas of p53(+/-) mice. Loss of heterozygosity studies of tumors indicated loss of heterozygosity in only 1 of 11 tumors. These data indicate clear differences between a p53 missense mutation and a null allele in tumorigenesis in vivo and suggest that the p53R172HDeltag mutant represents a gain-of-function allele.

Figure 1

The generation of a mouse containing a p53R172H substitution. (a) The genomic organization of the p53 gene and the targeting vectors are shown. The first recombination step results in incorporation of the neo and tk genes (PGKneoNTRtkpA) at the p53 locus. In the second step, the neo and tk genes are replaced with the genomic p53 containing a point mutation (asterisk), resulting in an arg-to-his substitution at amino acid 172. Δg represents the deletion of a G nucleotide at the intron 2 splice acceptor site. (b) Southern hybridization of EcoRI-digested DNA from several ES cell clones electroporated with the PGKneoNTRtkpA targeting vector. The 17-kb EcoRI fragment represents the wild-type p53 allele whereas the 3.4- and 12-kb EcoRI fragments are the expected sizes for the mutant. The ES cell clone (3C12) in lane 2 is correctly targeted. (c) SSCP analysis using primers spanning the mutation. Lanes: 1, plasmid containing wild-type p53 genomic sequences; 2, plasmid containing mutant p53 genomic sequences; 3, genomic DNA from targeted ES cell clone; 4, genomic DNA from normal ES cells. *, wild-type bands; **, mutant-specific bands. (d) Southern blot hybridization of EcoRI-digested DNA from derivatives of 3C12 cells targeted with a construct containing a missense mutation in p53. Lanes: 1, normal ES cell DNA; 2, 3C12 DNA; 3, DNA from a correctly targeted ES cell clone.

Figure 2

(a) Analysis of p53 protein levels in fibroblasts from embryos null for p53 (lane 1), wild-type (WT; lanes 2 and 3), or homozygous for the p53R172HΔg mutation (lane 4). Wild-type fibroblasts were treated with UV to show induction of p53 and confirm position on the gel (lane 3). (b) The structure of the p53 gene around exons 2–4 and the spliced products identified in fibroblasts from homozygous p53R172HΔg mutant mice. Δ denotes the G nucleotide in intron 2 that is deleted.

Figure 3

Comparisons of survival between p53 null mice and p53R172HΔg mice. The p53^−/− mice survival data set is from our lab, and the p53^+/− mice survival data set is from Harvey et al. (17). (a) Survival curves of 80 p53R172HΔg/R172HΔg mice and 40 p53^−/− mice. (b) Survival curves of 92 p53R172HΔg/+ mice and 150 p53^+/− mice.

Figure 4

Tumor latency of different tumor types from 56 tumors that developed in 51 p53R172HΔg/+ mice. Os, osteosarcomas; Fs, fibrosarcomas; As, angiosarcomas; Lp, lyposarcomas; Ad, adenocarcinomas; HC, hepatocellular carcinomas; Pa, poorly differentiated carcinomas; SC, squamous cell carcinomas; IlC, islet cell carcinomas; Ly, lymphomas; Aa, adenoma.

Figure 5

Frequency of metastasis from tumors of p53R172HΔg/+ mice. (a) Metastatic lesions from an osteosarcoma that developed in a p53R172HΔg/+ mouse. (A) Liver. (B) Lung. (b) Representative histologies of tumors and their metastatic spread. A, C, and E represent primary tumors; B, D, F, and G represent their corresponding metastases. (A and B) Hepatocelluar carcinomas with metastasis in lung. (C and D) Lung adenocarcinomas with metastasis in myocardium (E, F, and G) Osteosarcoma with metastasis in liver (F) and lung (G). (c) Frequency of metastasis from p53R172HΔg/+ mice vs. p53^+/− mice. p53^+/− metastasis data are from Harvey et al. (17) and Tervana et al. (18), respectively.

Figure 6

A loss of heterozygosity study of tumors from p53R172HΔg/+ mice. SSCP analysis was performed on tumor samples obtained by using laser capture technology. (a) LOH analysis of DNA from osteosarcomas from mice numbered 395 and 565 using A3 and A4 primers (see Materials and Methods). 565N indicates DNA isolated from normal tissue adjacent to tumor of mouse 565. (b) LOH analysis of DNA from a carcinoma from mouse 3124 using primers A3 and Mie5R (see Materials and Methods). Tail DNA from wild type (+/+), homozygous (Δg/Δg), and heterozygous (Δg/+) mice were used as controls to distinguish the two alleles.