Human tumor p53 mutations are selected for in mouse embryonic fibroblasts harboring a humanized p53 gene

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National Academy of Sciences

RESUMO

To date, there has been no way to examine induced human p53 gene mutations in cell cultures exposed to mutagenic factors, other than by restriction site analysis. Here, we used embryonic cells from our Hupki (human p53 knock-in) mouse strain to generate human p53 DNA-binding domain (DBD) mutations experimentally. Twenty cultures of untreated primary mouse Hupki fibroblasts and 20 short-wavelength UV light (UVC)-treated cultures (20J/m2) were passaged >20 times. Established Hupki embryonic fibroblast cell lines (HUFs) were genotyped by dideoxy DNA sequencing of p53 exons 4-9. Seven of the HUFs harbored point mutations in the humanized p53 DBD. Of the 9 mutations (6 single- and 1 triple-site mutation), 2 were at the most frequently mutated codons in human cancers (c.248 and c.273). The Affymetrix p53 GeneChip assay also readily identified the 6 single-base substitutions. All mutations in HUFs from UV-treated cultures were at dipyrimidine sites, including 3 nontranscribed strand C →T transitions. The mutant HUFs were deficient in p53 transactivation function, and missense mutants had high levels of nuclear p53 protein. In a second experiment, primary Hupki cells were exposed to the carcinogen aristolochic acid I (AAI). Five of 10 cultures that became established within 2 months harbored p53 DBD mutations. All were transversions, including 4 A → T substitutions on the nontranscribed strand, a hallmark of DNA mutation by AAI. We conclude that establishment of Hupki mouse fibroblasts in culture readily selects for p53 DBD mutations found in human tumors, providing a basis for generating experimental mutation patterns in human p53.

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