Mutagenesis of human p53 tumor suppressor gene sequences in embryonic fibroblasts of genetically-engineered mice.
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Biomedical subjects
Zhipei Liu
Publications and source records attributed to Zhipei Liu.
Making omelets without breaking eggs: E2F-mediated induction of cardiomyoycte cell proliferation without stimulation of apoptosis.
The fundamental role of E2F transcription factors in the regulation of proliferation is well established. According to a widely accepted model, E2F1, E2F2, and E2F3 are classified as "activating" E2Fs since they induce proliferation of quiescent cells whereas E2F4 and E2F5 do not have the power to incite cell cycle progression but are related to differentiation processes and were therefore considered to be "repressive". In addition, it has been postulated that "activating" E2Fs induce apoptosis in a wide variety of cell types depending on their expression level. However, we demonstrated recently that this 'threshold model' does not hold true for cardiomyocytes. In a series of experiments in which we overexpressed individual E2Fs we found that directed expression of E2F2, unlike E2F1, E2F3 and E2F5, did not induce apoptosis but even suppressed expression of several pro-apoptotic genes in primary cardiomyocytes. Furthermore, we established that not only E2F1, E2F2, and E2F3 but also E2F4 was able to induce S-phase entry of primary cardiomyocytes. Our results suggest that it is possible to utilize the proliferation-inducing properties of the E2Fs in cardiomyocytes without activation of potentially harmful pro-apoptotic traits. This finding might open a new access to stimulate regeneration in postmitotic tissues such as the heart.
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p53 mutations in benzo(a)pyrene-exposed human p53 knock-in murine fibroblasts correlate with p53 mutations in human lung tumors.
Human p53 mutation spectra differ significantly from one cancer type to another. One possible reason is that carcinogenic risk factors differ, and these factors elicit distinct mutation patterns. There has been no mammalian assay, however, with which to generate mutation patterns in human p53 sequences experimentally, hampering interpretation of the human tumor spectra. We have designed a new mammalian cell assay using gene targeting technology that selects and scores human p53 gene sequence mutations in human-p53 knock-in (Hupki) murine embryonic fibroblasts (HUF) that have undergone immortalization. With the Hupki assay we examined here whether benzo(a)pyrene (BaP), a major tobacco smoke carcinogen could elicit p53 mutation patterns characterizing the human lung tumor p53 mutation spectrum. We found that, in contrast to unexposed HUFs or HUFs exposed to other carcinogenic agents, HUFs exposed to BaP acquire mutations that display major features of the human lung tumor p53 mutation spectrum: (a) predominance of G-to-T mutations, (b) unequivocal strand bias of the transversions, and (c) a mutation hotspot at codons 157 to 158. These data are consistent with the hypothesis that BaP has a direct role in causing smokers' lung tumor p53 mutations. The assay can be used to examine various hypotheses on the endogenous or exogenous factors responsible for the p53 mutations in human tumors arising in other tissues.
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Human tumor p53 mutations are selected for in mouse embryonic fibroblasts harboring a humanized p53 gene.
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/m(2)) 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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Mouse models for generating P53 gene mutation spectra.
The p53 tumor suppressor gene lends itself to mutation spectra analysis, because the frequency of point mutations in human tumors is high, the locations of inactivating tumor mutations are numerous and dispersed, and all possible base substitutions are observed in human cancer. P53 tumor mutations induced experimentally in mice exposed to carcinogens have been described, but have not yet contributed significantly to our understanding of mutagenic mechanisms or of the origins of mutations in human cancers. Recently, gene-targeting technology has allowed development of a new mouse model, which explores experimentally the endogenous and environmental factors that may contribute to neoplastic disease in humans.
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