Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function

Department of Biology and Center for Cancer Research, and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 04/2002; 99(5):2948-53. DOI: 10.1073/pnas.052713099
Source: PubMed

ABSTRACT The p53 tumor suppressor gene is the most frequently mutated gene in human cancers, and germ-line p53 mutations cause a familial predisposition for cancer. Germ-line or sporadic p53 mutations are usually missense and typically affect the central DNA-binding domain of the protein. Because p53 functions as a tetrameric transcription factor, mutant p53 is thought to inhibit the function of wild-type p53 protein. Here, we studied the possible dominant-negative inhibition of wild-type p53 protein by two different, frequently occurring point mutations. The R270H and P275S mutations were targeted into the genome of mouse embryonic stem cells to allow the analysis of the effects of the mutant proteins expressed in normal cells at single-copy levels. In embryonic stem cells, the presence of a heterozygous point-mutated allele resulted in delayed transcriptional activation of several p53 downstream target genes on exposure to gamma irradiation. Doxorubicin-induced apoptosis was severely affected in the mutant embryonic stem cells compared with wild-type cells. Heterozygous mutant thymocytes had a severe defect in p53-dependent apoptotic pathways after treatment with gamma irradiation or doxorubicin, whereas p53-independent apoptotic pathways were intact. Together these data demonstrate that physiological expression of point-mutated p53 can strongly limit overall cellular p53 function, supporting the dominant-negative action of such mutants. Also, cells heterozygous for such mutations may be compromised in terms of tumor suppression and response to chemotherapeutic agents.

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    • "Those findings were also correlated to the decreased ability of WT TP53 to induce growth suppression and cell cycle arrest in the presence of TP53 mutant [Willis et al., 2004]. Furthermore, the dominant negative potential of two TP53 mutants (p.R270H and p.P275S, corresponding in humans to p.R273H and p.P278S, respectively) was also demonstrated in vivo, in mouse models [de Vries et al., 2002]. "
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    ABSTRACT: Loss-of-function, partial-function, altered-function, dominant-negative, temperature sensitive; interfering; contact; structural; unfolded; misfolded; dimeric; monomeric; non-cooperative; unstable; supertrans; superstable; intragenic suppressor. TP53 mutants: they are many, more than 2000 in fact, and they can be very diverse. Sporadic; germline; gain-of-function; oncogenic; rebel-angel; yin and yang; prion-like; metastasis-inducer; mediator of chemo-resistance; modifier of stemness. TP53 mutants can impact on important cancer clinical variables, in multiple, often subtle ways, as revealed by cell-based assays as well as animal models. Here we review studies investigating TP53 mutants for their effect on sequence-specific transactivation function, and especially recent findings on how TP53 mutants can exhibit gain-of-function properties. We also review reports on TP53 mutants' impact on cancer cell transcriptomes and studies with Li-Fraumeni patients trying to classify and predict phenotypes in relation to experimentally determined transcription fingerprints. Finally, we provide an example of the complexity of correlating TP53 mutant functionality to clinical variables in sporadic cancer patients. Conflicting results and limitations of experimental approaches notwithstanding, the study of TP53 mutants has provided a rich body of knowledge, mostly available in the public domain and accessible through databases, that is beginning to impact on cancer intervention strategies. This article is protected by copyright. All rights reserved.
    Human Mutation 06/2014; 35(6). DOI:10.1002/humu.22514 · 5.05 Impact Factor
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    • "The loxPflanked conditional STOP cassette (Tuveson et al., 2004) was cloned into the XhoI site in intron 1 of the murine Tp53 locus. The R270H missense mutation was generated as described previously (de Vries et al., 2002). In our colony, the presence of the loxP–STOPcassette–loxP (LSL) cassette in intron 1 of Tp53 genomic DNA is detected using the following primers to generate a wild-type band of 170 bp and a mutant band of 270 bp: wt F-5Ј- TTACACATCCAGCCTCTGTGG-3Ј; mutant F-5Ј-AGCTAG - CCACCATGGCTTGAGTAAGTCTGCA-3Ј; R-3Ј-CTTGGA - GACATAGCCACACTG-3Ј. "
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    ABSTRACT: Tp53 mutations are common in human prostate cancer (CaP), occurring with a frequency of ∼30% and ∼70% in localized and metastatic disease, respectively. In vitro studies have determined several common mutations of Tp53 that have specific gain-of-function properties in addition to loss of function, including the ability to promote castration-resistant (CR) growth of CaP cells in some contexts. To date, a lack of suitable mouse models has prohibited investigation of the role played by Tp53 mutations in mediating CaP progression in vivo. Here, we describe the effects of conditional expression of a mutant Tp53 (Tp53R270H; equivalent to the human hotspot mutant R273H) in the prostate epithelium of mice. Heterozygous “Tp53LSL-R270H/+” [129S4(Trp53tm3Tyj)] and “Nkx3.1-Cre” [129S(Nkx3-1tm3(cre)Mms)] mice with prostate-specific expression of the Tp53R270H mutation (p53R270H/+ Nkx3.1-Cre mice) were bred onto an FVB/N background via speed congenesis to produce strain FVB.129S4(Trp53tm3Tyj/wt); FVB.129S(Nkx3-1tm3(cre)Mms/wt) and littermate genotype negative control mice. These mutant mice had significantly increased incidences of prostatic intraepithelial neoplasia (PIN) lesions, and these appeared earlier, compared with the Nkx3.1 haploinsufficient (Nkx3.1-Cre het) littermate mice, which did not express the Tp53 mutation. PIN lesions in these mice showed consistent progression and some developed into invasive adenocarcinoma with a high grade, sarcomatoid or epithelial-mesenchymal transition (EMT) phenotype. PIN lesions were similar to those seen in PTEN conditional knockout mice, with evidence of AKT activation concomitant with neoplastic proliferation. However, the invasive tumor phenotype is rarely seen in previously described mouse models of prostatic neoplasia. These data indicate that the Tp53R270H mutation plays a role in CaP initiation. This finding has not previously been reported. Further characterization of this model, particularly in a setting of androgen deprivation, should allow further insight into the mechanisms by which the Tp53R270H mutation mediates CaP progression.
    Disease Models and Mechanisms 04/2012; 5(6). DOI:10.1242/dmm.008995 · 5.54 Impact Factor
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    • "Moreover, many mutations lead to an increased half-life of the otherwise rapidly degraded p53 protein and thereby to accumulation of this protein in cells [4]. Other tumor suppressor genes often lose their expression after mutation, but the point mutated p53 protein is often more stable and therefore overexpressed in tumor cells [5] [6]. p53 degradation can also be promoted directly through binding to viral proteins or deletions promoting presentation for T cell recognition [1] [2]. "
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    ABSTRACT: Approximately 50% of human malignancies carry p53 mutations, which makes it a potential antigenic target for cancer immunotherapy. Adoptive transfer with p53-specific cytotoxic T-lymphocytes (CTL) and CD4(+) T-helper cells eradicates p53-overexpressing tumors in mice. Furthermore, p53 antibodies and p53-specific CTLs can be detected in cancer patients, indicating that p53 is immunogenic. Based on these results, clinical trials were initiated. In this paper, we review immunological and clinical responses observed in cancer patients vaccinated with p53 targeting vaccines. In most trials, p53-specific vaccine-induced immunological responses were observed. Unfortunately, no clinical responses with significant reduction of tumor-burden have occurred. We will elaborate on possible explanations for this lack of clinical effectiveness. In the second part of this paper, we summarize several immunopotentiating combination strategies suitable for clinical use. In our opinion, future p53-vaccine studies should focus on addition of these immunopotentiating regimens to achieve clinically effective therapeutic vaccination strategies for cancer patients.
    BioMed Research International 03/2011; 2011:702146. DOI:10.1155/2011/702146 · 2.71 Impact Factor
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