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 p53R172H⌬g mutant represents a gain-of-function allele. M utations in the p53 tumor suppressor are common in the etiology of many tumors and disrupt the ability of p53 to induce growth arrest or apoptosis. In particular, the R175H mutation corresponds to one of six hot spot mutations in p53 and accounts for Ϸ6% of the missense mutations identified in human cancers (1).Mutant p53 proteins containing missense mutations have been studied in various assays, leading to suggestions that some may yield gain-of-function or dominant negative phenotypes. For example, expression of mutant p53 in p53 null cells yields tumors in syngeneic mice (2) and makes established cells highly tumorigenic (3). When assayed in comparison to other mutants, the p53R175H mutant was always the most detrimental (4-7). The p53R175H mutant cooperates better than any other mutant in ras-mediated transformation (4, 5). Cells expressing the p53R175H mutant are the most tumorigenic, as measured by growth in soft agar (6). In another experiment, genomic instability was measured in fibroblasts derived from Li-Fraumeni syndrome patients heterozygous for the p53R175H mutant (8). These cells exhibited a disrupted spindle checkpoint control and accumulated a polyploid DNA content as compared with cells with other p53 missense mutations, which arrest with a 4n DNA content. These data, coupled with the fact that most p53 mutations (Ͼ80%) occurring in human tumors are missense mutations, suggest that cells with p53 missense mutations have a growth advantage.These in vitro experiments, however, cannot assay the effect of mutant p53 on tumor growth rate, survival, and tumor spectrum in vivo. The generation of mice null for p53 (9, 10) has yielded a plethora of data as to the importance of p53 in vivo. Mice heterozygous or homozygous for a p53 null allele are predisposed to multiple tumor types such as lymphomas, sarcomas, and other tumors resembling to some extent the types of tumors seen in the Li-Fraumeni syndrome. Thymocytes from mice lacking p53...

Crippling p53 activities via knock-in mutations in mouse models

“…Moreover, p53 þ /R172H mice develop more osteosarcomas than p53 þ /-or p53 þ /R270H mice, and p53 þ /R270H mice produce carcinomas at higher frequency compared to other genotypes on the 129S 4 /SvJae background. p53 þ /R172HDg mice on a mixed C57BL/6-129SV background also show an increased number of carcinomas (Liu et al, 2000). In contrast, on the C57BL/6 background, no differences in tumor spectra are observed between p53 þ /R172H and p53 þ /-mice.…”

“…To address the physiological relevance of these missense mutations, several laboratories attempted to generate mouse models for three of the hot spot mutations: p53R172H (175 in human), p53R270H, (273 in human) and p53P275S (278 in human) (Liu et al, 2000;de Vries et al, 2002). These initial studies failed to recapitulate the human disease in mice.…”

“…In the p53R172H study, the targeted allele contains an additional deletion of a G nucleotide at a splice junction, leading to hypomorphic expression of the p53R172H mutant (this allele is referred to as p53 R172HDg ) (Liu et al, 2000). The hypomorphic nature of this mutation allowed chromosome stability in culture and generation of mice.…”

Chromosome stability, in the absence of apoptosis, is critical for suppression of tumorigenesis in Trp53 mutant mice

“…We cloned a PGK-neo r cassette flanked by two loxP sequences into the second AccI site of intron 4 of a Trp53 genomic fragment 28 . We generated the R172P substitution (CGC to CCC) at nucleotide 515 (taking the A in the first ATG as nucleotide number 1) in exon 5 of Trp53 by site-directed mutagenesis.…”

Mutant mouse p53 transgene elevates the chemical induction of tumors that respond to gene silencing with siRNA

“…Mice carrying p53 mutated at codon135 are highly sensitive to spontaneous 7,8 and chemical tumor induction. 9 Mice carrying p53 mutations analogous to the human Li-Fraumeni syndrome hot spot mutation exhibit a high incidence of spontaneous tumors with a spectrum different from that of p53-null mice, [10][11][12] a high metastatic potential for produced tumors 13 and a high incidence of chemically induced tumors. 14 Especially of note is an approach toward simulating the human context with heterozygous mice containing human p53 and Li-Fraumeni mutant p53 in the genome.…”