The high prevalence and great diversity of p53 tumor suppressor gene mutations in human tumors call for development of therapeutic molecules that rescue function of aberrant p53 protein. P53 mutations also offer new approaches to the study of the origins of mutations in human cancer. An experimental mouse model with a genetically modified but normal functioning p53 gene harboring the human rather than the murine core domain, would be of considerable benefit to research on both cancer therapeutics and etiology; however, it is uncertain whether such mice would permit biological functions of p53 to be retained. Using a Cre/lox P gene-targeting approach, we have constructed a human p53 knock-in (hupki) mouse strain in which exons 4–9 of the endogenous mouse p53 allele were replaced with the homologous, normal human p53 gene sequence. The chimeric p53 allele (p53KI) is properly spliced, transcribed in various tissues at levels equivalent to wild-type mice, and yields cDNA with the anticipated sequence, that is, with a core domain matching that of humans. The hupki p53 protein binds to p53 consensus sequences in gel mobility shift assays and accumulates in the nucleus of hupki fibroblasts in response to UV irradiation, as is characteristic of wild-type p53. Induction of various p53-regulated genes in spleen of γ-irradiated homozygous hupki mice (p53KI/KI), and the kinetics of p53-dependent apoptosis in thymocytes are similar to results with wild-type (p53+/+) mice, further indicating normal p53 pathway function in the hupki strain. The mice are phenotypically normal and do not develop spontaneous tumors at an early age, in contrast to knock-out (p53−/−) strains with a defective p53 gene. The chimeric (p53KI) allele thus appears to provide a biological equivalent to the endogenous murine (p53+) gene. This strain is a unique tool for examining in vivo spontaneous and induced mutations in human p53 gene sequences for comparison with published human tumor p53 mutation spectra. In addition, the hupki strain paves the way for mouse models in pre-clinical testing of pharmaceuticals designed to modulate DNA-binding activity of human p53.
The p53 protein is dysfunctional or absent in the majority of human tumors, primarily due to single missense point mutations in the gene (Vogelstein and Kinzler, 1992; Hainaut and Hollstein, 2000). Most of the 15 000 published human tumor mutations lie in the core (DNA-binding) domain (Harris and Hollstein, 1993; Lane, 1999), and reduce or abolish the ability of the p53 protein to bind to recognition sequences of p53-responsive genes and stimulate transcription. To investigate factors contributing to p53 tumor mutations in tumors, published p53 mutation spectra of human cancers can be compared with experimentally induced tumor p53 mutations in standard laboratory mouse strains (reviewed in Hollstein et al., 1999); however, there has been to date no practical approach to the experimental induction of mutations in the human p53 gene sequence in vivo. The core domains of the mouse and human p53 genes differ at 15% of base residues. Since mutation patterns are dictated in the first instance by the exact DNA base context, even subtle sequence differences can modify mutation spectra. Expectedly, specific carcinogen-induced p53 tumor mutations do not accumulate at precisely the same sites in the mouse and in humans (Dumaz et al., 1997). These considerations call for generation of mouse strain with human p53 sequences, which would be particularly relevant if the modified gene were transcribed at normal levels and encoded a functional protein.
A human p53 knock-in mouse strain of this nature would also lend itself to investigation of molecules designed to target the tumor suppressor protein. While mice with an unmodified endogenous p53 gene can be used to test the efficacy of drugs targeting the mouse p53 protein, human/mouse species sequence differences complicate assessment of experimental results for humans. Although the similarity between mouse and human p53 is remarkably high at the amino acid level (91% homology in the core domain), single residue differences may affect response to drugs designed either to restore DNA-binding activity of the common human tumor missense mutants (Foster et al., 1999), or conversely, to block wild-type protein function temporarily in order to reduce side effects of chemo- or radiation therapy (Komarov et al., 1999). Genetically engineered mice with a ‘humanized’ p53 gene thus would provide a new dimension to existing in vivo test systems not only for mutagenesis studies but also for pre-clinical testing of p53-modifying drugs; however, it remains to be shown whether in this chimeric context, p53 would continue to exercise its cellular activities.
Towards this end, we generated a mouse strain with p53 gene harboring the human core domain sequence. In these mice, referred to as hupki mice (for human p53 knock-in), exons 4–9 and intervening introns of the mouse allele have been replaced with the corresponding homologous segment of the normal human p53 gene, and transcription remains under endogenous control of the mouse locus. Biochemical and functional studies described here demonstrate that the genetically-modified p53 gene of homozygous hupki mice is expressed at physiological levels and can perform various cellular functions of normal p53.
Generation of human p53 knock-in (hupki) mice
After identification of ES clones containing the recombinant allele (p53KI) of p53 (Figure 1, upper panel), ES cells were injected into blastocytes to generate chimeric mice. Germline transmission of the knock-in allele originally derived from ES clone 3–16 was identified by Southern blotting after breeding chimeric mice with wild-type mice (Figure 1). F1 germline heterozygous offspring (p53KI/+) were intercrossed to produce F2 homozygous hupki mice. Both heterozygous and homozygous hupki mice from such crosses were born at the expected frequency (Mendelian ratio), and homozygous hupki mice are phenotypically normal and fertile (data not shown), suggesting that the chimeric p53 protein is compatible with mouse development. Homozygous hupki mice developed no spontaneous tumors by 8 months of age, in contrast to p53 null mice which are highly susceptible to lymphomas and sarcomas at an early age (Donehower et al., 1992; Jacks et al., 1994).
To examine whether the chimeric p53 is transcribed correctly in vivo, p53 cDNA prepared from hupki (p53KI/KI) mouse spleen RNA was sequenced and shown to match precisely the mouse wild-type p53 gene sequence in exons 2, 3, 10 and 11, whereas exons 4–9 (codons 33–332) were identical to the normal human p53 sequence, as expected. The human polymorphic codon 72 encodes arginine in the p53KI gene. The cDNA sequence confirmed that the targeted chimeric gene is correctly spliced and transcribed in the spleen of hupki mice (data not shown).
Analysis of p53KI gene expression
Our objective was to generate a mouse with a human/mouse chimeric p53 gene at the endogenous locus that would remain under normal transcriptional regulation, and would maintain normal p53 tumor suppressor-associated functions. In view of the high divergence between mouse and human intron sequences, and their possible role in transcription regulation (Shamser and Montano, 1996), we first compared p53 mRNA levels in various tissues of 10-week-old wild-type (p53+/+) and p53KI/KI mice, and explored possible transcription preferences of p53+ and p53KI alleles by three different approaches: (i) semiquantitative RT–PCR: Duplex semiquantitative RT–PCR of RNA extracted from 10 hupki (p53KI/KI) and wild-type (p53+/+) tissues using β-actin (reference sequence) and p53- specific primer pairs indicated similar levels of p53 expression for the two genotypes in each tissue compared (Figure 2A); p53 expression is high in spleen, thymus, lung and kidney, relative to levels in brain; (ii) restriction patterns of p53 RT–PCR products: To corroborate that the p53KI transcript is present in various tissues and at levels comparable to wild-type mice, we took advantage of AvaI restriction sites present in exon 6 of the normal mouse p53 gene, but absent in the human exon 6 sequence. RT–PCR amplifications encompassing exon 6 and using template from p53KI/+ heterozygotes, followed by enzymatic digestion with AvaI yielded comparable amounts of cleaved product (from the p53+ allele) and uncleaved product (from the p53KI allele) for all 10 organs examined. (Results with liver and spleen are shown in Figure 2B; data not presented for the eight additional tissues referred to in Figure 2A); and (iii) Northern blotting: The presence of equivalent p53 mRNA levels in p53KI/KI and p53+/+ mice was verified in thymus, spleen and brain (showing high, medium and low levels respectively) of 10-week-old mice by Northern blot analysis using exon 11 probe P (data not shown).
The presence of hupki p53 protein, and its accumulation following DNA damage by γ-irradiation were examined on immunoblots from primary embryonic fibroblast whole cell extracts using anti-mouse p53 polyclonal antibody CM5. As shown in Figure 3A, p53KI protein from hupki cells is expressed, and levels increase following 5 Gy γ-irradiation, as is seen with wild-type murine p53 (p53+). With CM5 antiserum, p53 signal intensity on immunoblots is lower in hupki and in human neonatal fibroblast extracts than in extracts from wild-type mice (data not shown), as anticipated from Pepscan ELISA analysis of CM5 (Lane et al., 1996), so the issue of whether p53 protein levels are equivalent in wild-type and hupki cells is not addressed in this experiment. Electromobility shift assays (EMSA) with protein from irradiated primary embryonic fibroblasts were performed to test whether the hupki protein is able to bind to p53 target DNA sequences, a pre-requisite for many tumor suppressor activities of p53. Experiments using p53 consensus oligonucleotide p53CON and anti-p53 antibody PAb421 revealed specific complexes with hupki cell extracts, as was observed with complexes from wild-type cell extracts (Figure 3B). Specific competition with non-radioactive consensus sequence abolished the signal, whereas a 20-fold excess of mutant consensus sequence, which does not bind p53 protein had no effect on signal intensity (Figure 3B).
P53 functional analysis
Transcriptional regulation of numerous genes is critical to p53-mediated cell cycle arrest and apoptosis following DNA damage by γ-irradiation. Specific transactivation of two well-studied genes, p21/Waf1/Cip1, encoding a cyclin-dependent kinase, and Bax, a regulator of programmed cell death, was initially observed by semi-quantitative RT–PCR in spleen of γ-irradiated (5 Gy) wild-type and homozygous hupki mice 6 and 24 h post treatment (data not shown). Irradiation induction of Bax and p21/Waf1 was verified in both genotypes by Northern blot analysis (Figure 4A). Gene expression profiling was performed with Clontech Mouse 1.2 AtlasTM membranes (>1000 distinct cDNA sequences) for spleen of homozygous hupki mice to investigate concordance with wild-type profiles. Expression patterns were highly similar in the two genotypes, both from untreated and from irradiated mice 24 h post treatment; Hupki spleen transcript levels of numerous genes involved in distinct cellular processes were induced by treatment, including p53-inducible genes Cyclin G, and Mdm2, consistent with expectations from the literature on p53 wild-type mice (Table 1).
Wild-type p53 protein in normal cells accumulates in nuclei in response to ultraviolet light (UV)-induced DNA damage to levels readily detected by immunocytochemistry. We investigated this response to UV irradiation in hupki cells. Nuclei of hupki (p53KI/KI) embryonic fibroblasts exposed 24 h previously to25 J/m2 UV light (254 nm) revealed intense nuclear staining with polyclonal p53 antibody CM1, whereas nuclei of unirradiated control cell cultures showed faint to no signal with the same staining procedure (Figure 4b). This response is further indication that the hupki p53 protein is subject to this cellular regulation in a manner similar to wild-type p53.
P53-dependent and -independent apoptosis in thymocytes of hupki mice
To test whether the chimeric p53 protein would function in a p53-dependent biological process, we studied apoptosis induced by DNA damaging agents. Thymocytes were isolated from wild-type (p53+/+), hupki (p53KI/KI), and p53 null (p53−/−) animals, and exposed to ionizing radiation (Figure 5), which induces apoptosis that is dependent on functional p53, or to dexamethasone, which induces p53-independent apoptosis in this cell type. After treatment with dexamethasone, thymocytes from all three genotypes exhibited apoptosis with similar kinetics (Figure 5A), indicating that this death pathway was not affected by the absence of wild-type p53 or the presence of hupki p53 protein. As expected, treatment with 5 Gy γ-irradiation induced apoptosis in wild-type cells, whereas thymocytes from p53−/− mice were profoundly resistant to the effects of ionizing radiation (Figure 5B), consistent with previously published results (Lowe et al., 1993; Clarke et al., 1993). Notably, thymocytes from hupki mice were susceptible to γ-irradiation-induced apoptosis at the same levels as those in wild-type thymocytes. In addition, in a dose-response experiment, hupki thymocytes exhibited apoptosis after various doses of γ-ray treatment with similar kinetics to wild-type cells at all doses tested (Figure 5C). These data indicate that the chimeric hupki p53 protein functions as efficiently as wild-type p53 with respect to its role in thymocyte apoptosis.
We have generated a mouse strain in which human p53 gene sequences encompassing the p53 core domain are embedded in the endogenous p53 locus on mouse chromosome 11, and replace the corresponding mouse gene segment. We designed the targeting vector so that the entire core domain and flanking sequences would be homogeneous with respect to species origin, since an engineered human/murine chimeric core domain theoretically could present a sequence configuration that would render various subsequent somatic missense mutations innocuous by an intragenic suppressor mechanism (Brachmann et al., 1998). Expression and endogenous regulation of the modified p53 gene (p53KI) have been achieved in homozygous hupki mice, despite the chimeric nature of the allele and the notorious sensitivity of p53 to minor sequence alterations not directed by evolution (Hainaut and Hollstein, 2000; Brachmann et al., 1998; Cho et al., 1994).
It has been shown recently that arginine at the common Arg/Pro polymorphic residue 72 of human p53 can enhance tumorigenicity of certain p53 mutants by compromising control of apoptosis by p73β, a binding partner of p53 (Marin et al., 2000). Since the hupki protein harbors the Arg72 residue, this raises the interesting possibility that presence of this residue will recapitulate the human situation by increasing tumorigenic potential of somatic mutations arising in mutagen-exposed hupki mice.
The biochemical and functional studies we have conducted thus far indicate we have achieved our aim to generate human p53 knock-in mice that retain in vivo physiological control of p53 and several of its functions. The hupki p53KI/KI mice exhibit normal development, fertility, and a p53 phenotype thus far corresponding to wild-type p53+/+ mice of the same genetic background. Expression profiling with AffymetrixTM gene expression arrays is underway to explore potential differences between hupki and normal mice in the complex transcription patterns/induction kinetics of p53-downstream genes (Yu et al., 1999). Recent studies have demonstrated that distinct subsets of genes are regulated by p53 according to the nature of the inducing factor and cell type (Yu et al., 1999; Zhao et al., 2000). We chose spleen tissue, γ-irradiation, and 6 or 24 h post-treatment times for initial characterization of hupki mice; however, since a complexity of phosphorylation events and protein–protein interactions govern p53 function (Prives and Hall, 1999), further investigations using other parameters may reveal more subtle differences between hupki and wild-type mice in post-translational modification, biological function, or kinetics of activation. An important aspect of p53 function to address in future experiments is the ability of hupki p53 protein to perform similarly to wild-type p53 as participant in processes of DNA repair and recombination (Albrechtsen et al., 1999; Dudenhoffer et al., 1999). Paradoxically, a hupki mouse deficiency in p53 ‘guardian of the genome’ activities could enhance fortuitously the usefulness of this mouse model by rendering the strain more susceptible to tumorigenesis when exposed to carcinogenic DNA damaging agents.
In principle, hypotheses regarding mutation induction by specific cancer risk factors may be tested by comparing p53 tumor mutations typical of a defined high-risk patient group with mutations generated experimentally in rodents, or even in simple cell systems such as yeast (Hainaut and Hollstein, 2000, Dumaz et al., 1997; Flaman et al., 1994). In practice, fundamental questions remain unanswered that require additional experimental models: In vivo ‘spontaneous’ p53 mutation patterns in humans in different cell/tissue types, in inflamed tissues, in regenerating tissues following toxicity or injury, or induced by oxidative stress, are ill-defined (Hollstein et al,. 1998; Beckman and Ames, 1998). These are key issues, since for a number of common cancers it is unlikely that chemical carcinogens are a major contributor to the tumor p53 mutations observed (Hollstein et al., 1998; Baker et al., 1991).
The hupki mouse offers a new refinement to p53 mutagenesis studies in rodents because it provides an in vivo rodent test model, yet with the precise human p53 sequence as mutation target. Various applications are open to investigators, such as detection of unselected mutations in normal tissues using sensitive PCR-based protocols (Aguilar et al., 1993), mapping of DNA damage sites in the p53 gene to show co-localization of damage sites with human tumor mutation hotspots in the p53 gene (Denissenko et al., 1996), or screening of neoplastic lesions and carcinomas in hupki mice for p53 core domain mutations with the Affymetrix p53GeneChipTM oligonucleotide array, an automated method applicable to hupki mouse samples because the p53 sequence is identical to the human sequence in exons 4–9. Tumor mutation spectra from experiments in hupki mice can be compared directly to the published human tumor p53 mutation data, unlike p53 mutations profiles that have been generated from examining tumors in wild-type mice.
We anticipate that the hupki strain or its derivatives will pave the way for pre-clinical in vivo testing of new pharmaceuticals designed to modulate human p53 tumor suppressor DNA binding/transcription activation functions. The hupki strain facilitates further genetic refinement involving, for example, recombinational exchange of amino or carboxy terminal p53 sequences, which also are of keen interest in the development of new therapeutic strategies (Hupp et al., 1995; Selivanova et al., 1997; Rodriguez et al., 1999). Hupki strain derivatives carrying single point mutations discovered in Li-Fraumeni syndrome cancer-prone families (Kleihues et al., 1997) would provide a new mouse model with an allelic configuration that parallels the human situation, and complement existing animal models with mutant mouse p53 transgenes or p53-deficient genotypes (Donehower et al., 1992; Jacks et al., 1994; Hurstings et al., 1995; Zhang et al., 2000; Liu et al., 2000).
Materials and methods
Construction of the gene-targeting vector
DNA fragments A, C, and D of the gene targeting vector (Figure 1, upper panel) were obtained as follows: Fragments A′ and D were cloned from a 129/Sv genomic library. PCR-generated fragments (A, C), from template A′ and normal human genomic DNA respectively, were cloned into pGEM (Promega) vectors and sequenced using Big DyeTM (Applied Biosystems International, ABI) dideoxy chain terminators and an ABI Gene Analyzer Model 310 to confirm that no mutations were introduced by Pfu polymerase into coding or splice site sequences. Fragment B, comprising loxP-flanked HSV-thymidine kinase (TK) and neomycin phosphotransferase (neo) gene sequences, was obtained from XbaI/HindIII digestion of plasmid pHR-1. Fragments A–D then were introduced step-wise into a pBluescript KS II (Stratagene) plasmid to generate the targeting vector (Figure 1, upper panel).
Gene targeting in embryonic stem (ES) cells and generation of hupki mice
After electroporation of the targeting vector into ES cells (E14.1) and selection, ES clones with homologous recombination events at the endogenous p53 locus were identified by both PCR and Southern blotting analysis (Figure 1, lower panel). The correctly targeted ES clones were subjected to transfection with a Cre-expressing vector (pMC-Cre) which deletes the neomycin/TK cassette flanked by loxP sites to generate the knock-in allele (abbreviated KI in Figures 1–5). Clones with recombinant mouse–human p53 sequences were identified, total RNA was isolated, and the p53 cDNA sequence verified by dideoxy sequencing. Mouse-specific primers for sequencing, PCR or RT–PCR are from Ushijima et al. (1995) unless otherwise indicated. Sequences of primers for human p53 segments are from Lehman et al. (1991) and from the Affymetrix p53 GeneChipTM protocol. ES clones were injected into blastocytes to generate chimeric mice. Germline offspring were identified by Southern blotting analysis after breeding of chimeric mice with C57/BL/6 or 129/Sv wild-type mice (Figure 1, lower panel). All experiments were performed using mice of mixed genetic background (129/Sv–C57BL/6).
Isolation and culture of primary mouse embryonic fibroblasts (MEFs)
Embryonic fibroblasts were isolated from embryos of intercrosses of p53KI/+ mice at E13.5 mid-gestation and cultured as previously described (Wang et al., 1995): Fetuses were dissected from uteri and digested five times, 5 min each, with 0.25% trypsin (Gibco-BRL) at 37°C. Cells were then transferred to tissue culture flasks and cultured in standard DMEM medium supplemented with 10% fetal calf serum. Experiments were carried out with primary fibroblasts of each genotype (passages 2–3), derived from embryos of the same litter.
RNA analysis by semiquantitative RT–PCR, Northern blots, and Clontech cDNA arrays
Total RNA was extracted for RT–PCR or Northern analysis with an RNeasy Kit (Qiagen) according to supplier's instructions. First strand cDNA synthesis was accomplished with a PromegaTM RT Kit. Duplex RT–PCR of tissue RNA with primers specific for p53 (P42A, P52A, P10B, Ushijima et al., 1995), p21/Waf1 (5′-CGGTCCCG TGGACAGTGAGC-3′; 5′-AAATCTGTCAGGC TGGTCTGCC-3′), Bax (5′-GCGTCCA CCAAGAAGCTGAG-3′; 5′-CCAC CCTGGTCTTGGATCCA-3′), β-tubulin (5′-GACAGTGTGGCAACCAGATCG-3′, 5′-GTACGGAAGCAGATGTCGTAG-3′) and β-actin (5′-TGTGATGGTGGGAATGGGTCAG-3′; 5′-TTTGATGTCACGCACGATTTC-3′) was performed initially in 50 μl reaction volumes over a range of cycles (20–35) in a thermal cycler using the following cycling conditions: 95°C, 1 min; 60°C, 1 min; 72°C, 45 s. For Northern analysis, 10–15 μg total RNA per lane were electrophoresed on formaldehyde-containing gels, transferred by capillary blot onto GeneScreenPlusTM (DuPont) nylon membranes and hybridized to specific probes; probe P was generated by PCR with 129/Sv mouse genomic DNA template and primers P11A (5′-ACCAAGAAGGGCCAGTCTAC-3′) and P11B (5′-TGGAGGATATGG ACCCTATG-3′); Bax-, p21- or β-actin (control) probes were generated using primers as given above for RT–PCR. Washed blots were exposed to Amersham X-ray film for 1–7 days. cDNA Arrays: Procedures for obtaining RNA, cDNA and hybridization protocols for Clontech AtlasTM Mouse 1.2 Microarrays were as recommended by the manufacturer. Hybridized arrays were exposed to X-ray film (32P) for 3–7 days, or to FujiImageTM plates (33P) for PhosphorImager visualization. Data were evaluated by ArrayVisionTM software (Imaging Research Inc., Canada).
Protein analysis by immunoblot and electromobility shift assay (EMSA)
P53 consensus sequence p53CON, and p53 monoclonal antibody PAb421 were purchased from Santa Cruz Biotechnology. The assay was performed according to supplier's instructions, with 20 μg whole cell extracts from γ-irradiated (5 Gy) fibroblasts, 6 h post-treatment. Unlabeled p53CON (20–) were used as specific competitor, and the p53 mutant oligonucleotide (20–) (Santa Cruz) as non-specific competitor. Proteins for immunoblots were extracted from embryonic fibroblasts in RIPA buffer (25 mM Tris pH 8.2, 50 mM NaCl, 0.1% SDS, 0.5% Nonidet P-40, 0.5% deoxycholate) containing CompleteTM proteinase inhibitor cocktail (Boehringer Mannheim), followed by centrifugation through Qiagen ‘shredders’. Twelve μg protein per lane were loaded onto SDS–PAGE gels, electrophoresed, and gels were blotted onto PVDF membranes. Primary antibodies or antisera used were CM5 (anti-p53, Novacastra, Newcastle upon Tyne, UK) or anti-laminin-β1 (C-19, Santa Cruz Biotechnology, CA, USA). Protein from the AP43a (p53−/−) mouse embryo fibroblast cell line (neg. control), and from neonatal primary human foreskin fibroblasts (positive control) were examined in parallel.
Irradiation of whole mice and embryonic fibroblasts
Whole body γ-irradiation (5 Gy) was performed on 10-week-old p53+/+, p53KI/+ and p53KI/KI mice. Mice were sacrificed 6 or 24 h post-irradiation. Fibroblasts from embryos were obtained according to our previously published protocol (Wang et al., 1995). Primary embryonic fibroblast monolayers were irradiated (5 Gy), and cultured an additional 6 or 24 h before extraction of RNA or protein. Embryonic fibroblast cell line MEF 42(B1) derived from p53KI/KI embryos was irradiated with 25 J/m2 in a Stratagene StratalinkerTM and examined for p53 protein nuclear accumulation at 24 h post treatment by immunocytochemistry with CM1 antiserum.
Apoptosis in thymocytes treated with DNA damaging agents
Thymocytes were isolated from wild-type, hupki, and p53 null mice at 6 weeks of age as previously described (Wang et al., 1995), and adjusted to a density of 1–106 cells per ml. At time zero, cultures were treated (see legend to Figure 5), distributed into 35 mm wells (1–106 per well) and incubated at 37°C. Apoptotic cells were stained with ApoAlert Annexin V Kit (Clontech, Palo Alto, CA, USA) reagents, and analysed with FACScan (Becton Dickinson).
- hupki :
human p53 knock-in mice
Aguilar F, Hussain SP and Cerutti P. . 1993 Proc. Natl. Acad. Sci. USA 90: 8586–8590.
Albrechtsen N, Dornreiter I, Grosse F, Kim E, Wiesmuller L and Deppert W. . 1999 Oncogene 18: 7706–7717.
Baker SJ, Preisinger AC, Jessup JM, Paraskeva C, Markowitz S, Willson JKV, Hamilton S and Vogelstein B. . 1991 Cancer Res. 50: 7717–7722.
Beckman KB and Ames BN. . 1998 Physiol. Rev. 78: 547–581.
Brachmann RK, Yu K, Eby Y, Pavletich NP and Boeke JD. . 1998 EMBO J. 17: 1847–1859.
Cho Y, Gorina S, Jeffrey PD and Pavletich NP. . 1994 Science 265: 346–355.
Clarke AR, Purdie CA, Harrison DJ, Morris RG, Bird CC, Hooper ML and Wyllie AH. . 1993 Nature 362: 849–852.
Denissenko MF, Pao A, Tang M and Pfeifer GP. . 1996 Science 274: 430–432.
Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA, Butel JS and Bradley A. . 1992 Nature 356: 215–221.
Dudenhoffer C, Kurth M, Janus F, Deppert W and Wiesmuller L. . 1999 Oncogene 18: 5773–5784.
Dumaz N, van Kranen HJ, de Vries A, Berg RJW, Wester PW, van Kreijl CF, Sarasin A, Daya-Grosjean L and deGruijl FR. . 1997 Carcinogenesis 18: 897–904.
Flaman JM, Frebourg T, Moreau F, Charbonnier C, Martin C, Ishioka C, Friend SH & Iggo R. . 1994 Nucleic Acids Res. 22: 3259–3260.
Foster BA, Coffey HA, Morin MJ, Rastinejad F. . 1999 Science 286: 2507–2510.
Hainaut P and Hollstein M. . 2000 Adv. Cancer Res. 77: 81–137.
Harris CC and Hollstein M. . 1993 New Engl. J. Med. 329: 1318–1327.
Hollstein M, Hergenhahn M, Yang Q, Bartsch H, Wang Z-Q and Hainaut P. . 1999 Mutat. Res. 431: 199–209.
Hollstein M, Moeckel G, Hergenhahn M, Spiegelhalder B, Keil M, Werle-Schneider G, Bartsch H and Brickman J. . 1998 Mutation Res. 405: 145–154.
Hupp TR, Sparks Z and Lane DP. . 1995 Cell 83: 237–245.
Hurstings SD, Perkins SN, Haines DC, Ward JM and Phang JM. . 1995 Cancer Res. 55: 3949–3953.
Jacks T, Remington BO, Williams EM, Schmitt S, Halachmi S, Bronson RT and Weinberg RA. . 1994 Curr. Biol. 4: 1–7.
Kleihues P, Schaeuble B, zur Hausen A, Esteve J and Ohgaki H. . 1997 Am. J. Pathology 150: 1–13.
Komarov PG, Komarova EA, Kondratov RV, Christov-Tselkov K, Coon JS, Chernov, MV and Gudkov AW. . 1999 Science 285: 1733–1737.
Lane DP. . 1999 Br. J. Cancer 80: 1–5.
Lane DP, Stephen CW, Midgley CA, Sparks A, Hupp TR, Daniels DA, Greaves R, Reid A, Vojtesek B and Picksley SM. . 1996 Oncogene 12: 2461–2466.
Lehman TA, Bennett WP, Metcalf RA, Welsh JA, Ecker J, Modali R, Ullrich S, Romano JW, Appella E, Testa JR, Gerwin BI and Harris CC. . 1991 Cancer Res. 51: 4090–4096.
Liu G, McDonnell TJ, Montes de Oca Luna R, Kapoor M, Mims B, El-Naggar AK and Lozano G. . 2000 Proc. Natl. Acad. Sci. USA 97: 4174–4179.
Lowe SW, Schmitt EM, Smith SW, Osborne BA and Jacks T. . 1993 Nature 362: 847–852.
Marin MC, Jost CA, Brooks LA, Irwin MS, O'Nions J, Tidy JA, James N, McGregor JM, Harwood CA, Yulug IG, Vousden KH, Allday MJ, Gusterson B, Ikawa S, Hinds PW, Crook T and Kaelin WG. . 2000 Nature Genetics 25: 47–53.
Prives C and Hall PA. . 1999 J. Pathol. 187: 112–126.
Rodriguez MS, Desterro JMP, Lain S, Midgley CA, Lane DP and Hay RT. . 1999 EMBO J. 18: 6455–6461.
Selivanova G, Iotosova WV, Okan I, Fritsche M, Strom M, Groner B, Grafstrom RC and Wiman KG. . (1997). Nature Medicine 3: 632–638.
Shamser M and Montano X. . 1996 Gene 176: 259–262.
Ushijima T, Makino H, Okonogi H, Hosoya Y, Sugimura T and Nagao M. . 1995 Mol. Carcinogenesis 12: 23–30.
Vogelstein B and Kinzler KW. . 1992 Cell 70: 523–526.
Wang ZQ, Auer B, Stingl L, Berghammer H, Haidacher D, Schweiger M and Wagner EF. . 1995 Genes and Dev. 9: 509–520.
Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW and Vogelstein B. . 1999 Proc. Natl. Acad. Sci. USA 96: 14517–14522.
Zhang Z, Liu Q, Lantry LE, Wang Y, Kelloff GJ, Anderson MW, Wiseman RW, Lubet RA and You M. . 2000 Cancer Res. 60: 901–907.
Zhao R, Gish K, Murphy M, Yin Y, Notterman D, Hoffman WH, Tom E, Mack DH and Levine AJ. . 2000 Genes and Development 14: 981–993.
This study was supported by grant number R01CA79493 from the National Cancer Institute (NCI, USA). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute (NCI). We are grateful to Dr R Koomagi for immunocytochemistry studies, and thank U Schmitt, K-R Muehlbauer, Joslyn Michelon and Dominique Galendo for technical assistance.
About this article
Nature Communications (2019)
Scientific Reports (2015)
Cell Death & Differentiation (2013)
Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells
Nature Protocols (2012)
Nature Reviews Genetics (2012)