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First published online 13 May 2008
doi: 10.1242/jcs.022822

Journal of Cell Science 121, 1899-1906 (2008)
Published by The Company of Biologists 2008
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The R246S hot-spot p53 mutant exerts dominant-negative effects in embryonic stem cells in vitro and in vivo

Ming Kei Lee1 and Kanaga Sabapathy1,2,*

1 Division of Cellular and Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610, Singapore
2 Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore


Figure 1
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  		Fig. 1. Generation and characterization of R246S knock-in ES cells. (A) Schematic of the targeting strategy. Black boxes represent p53 exons (exon numbers are indicated above). The positions of the probes used for Southern blotting are indicated below the diagram. Floxed neomycin and DTA expression cassettes are also indicated. Restriction sites are indicated as follows: E, H, Bs and B represents EcoRI, HindIII, BssHII and BamHI, respectively. Expected fragment sizes for Southern screening are indicted by the arrows and the position of the R246S mutation is indicated with the arrowhead. WT, wild type; Rec, after Cre-mediated recombination. The small green triangle represents the single loxP site remaining after Cre-mediated recombination. (B) Results of Southern blot hybridization, using exon 11 as the internal probe (left panel) and exon 1 as the external probe (right panel), after EcoRI digestion are shown for one representative targeted clone (p53+/R246S-Neo) and one clone with the targeted- and neomycin-cassette removed (p53+/R246S). Arrowheads point to the position of the expected bands for the mutant allele. (C) Expression of the mutant p53 allele in ES cells was analyzed by RT-PCR-RFLP. ES cells of various genotypes were treated with 0.5 µg/ml doxorubicin (Dox.) for 3 hours and p53 transcripts were PCR amplified prior to digestion with BsrBI. Restriction fragments generated from the respective alleles are indicated with arrowheads. (D) Conformation of R246S mutant p53 protein was determined by immunoprecipitation using conformation-specific antibodies Pab240, which recognizes the mutant, and Pab246, which recognizes the wild-type conformation, followed by immunoblotting. H1299 cells expressing the R175H human p53 mutant were used as a positive control for mutant conformation. (E) Protein level of p53 and actin was determined by western blotting. (F) Localization of p53 was analyzed by fluorescence confocal microscopy. ES cells were mock (–{gamma}) or 20-Gy (+{gamma})-irradiated and harvested 3 hours later. Representative images are shown in which the green fluorescence represents p53 protein and red fluorescence represents genomic DNA.

 

Figure 2
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  		Fig. 2. R246S mutant protein exhibits DN effects in ES cells. (A) The expression of p53 target genes noxa, p21 and mdm2 was analyzed by quantitative real-time PCR 6 hours after doxorubicin (0.5 µg/ml) treatment. The expression level of each gene was normalized with the expression of gapdh and fold induction was calculated. Data represents mean ± s.e.m. of four independent experiments, each performed in duplicate. (B) Expression of mdm2 mRNA was analyzed by northern blot hybridization after doxorubicin (0.5 µg/ml) or {gamma}-irradiation (5 Gy). Intensity of 28S ribosomal RNA on agarose gel is shown for the loading control. (C,D) Cell-death analysis upon genotoxic stresses. Undifferentiated ES cells were treated with various doses of doxorubicin (C) or UV (D) for 12 hours and cell death was determined by flow cytometry after staining with annexin-V/propidium iodide. Data represents the percentage survival normalized to untreated samples (mean ± s.e.m.) from four independent experiments, each performed in duplicate.

 

Figure 3
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  		Fig. 3. R246S mutant protein does not affect differentiation of ES cells but also exerts DN effects in the differentiated state. (A) Pluripotency of ES cells is not affected by expression of R246S mutant p53. The expression of various stem-cell markers was analyzed by semi-quantitative RT-PCR (left panel). The morphology of the undifferentiated ES-cell colonies was routinely monitored by phase-contrast microscopy and representative images of ES cells of various p53 genotypes are shown (right panel). (B) The expression of stem-cell markers was analyzed during retinoic-acid-induced (0.1 µM) differentiation of ES cells. (C) Localization of p53 in ES cells differentiated with 0.1 µM retinoic acid for 6 days, after treatment with (+{gamma}) or without (–{gamma}) 20 Gy irradiation, was analyzed by fluorescence confocal microscopy. The green fluorescence represents p53 protein; red fluorescence represents genomic DNA. (D,E) Differentiated ES cells were irradiated with 20 Gy and harvested 24 hours later. (D) Cell-cycle analysis was performed by flow cytometry and ModFit cell cycle analysis software. (E) Cellular proliferation was determined by BrdU staining of cells (treated without or with doxorubicin) followed by flow-cytometric analysis. Data represents the percentage reduction of S-phase cells (D) or the percentage of BrdU+ cells (E) (mean ± s.e.m.) from three independent experiments, each performed in duplicate.

 

Figure 4
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  		Fig. 4. p53+/R246S ES cells are as highly tumorigenic as the p53–/– cells. (A) Representative images of livers from scid mice injected with ES cells of various p53 genotypes. Four mice per group were used, all of which gave consistent results. (B) Fluorescence microscopy of ES-cell-injected livers before fixation. Green fluorescence indicates the expression of GFP in the tumour nodules in livers. (C) Histological analysis of livers injected with ES cells of various p53 genotypes. The tumour portions derived from the differentiated ES cells are highlighted by dotted lines. Normal liver is indicated by `Li', whereas `B', `C', `E', `F' and `M' indicate the bone, cartilage, epithelium, fibroblast-like cells and smooth muscles, respectively.

 

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