Introduction of a p53.S389A point mutation into ES cells and mice. (A) Scheme for targeting one allele of the murine p53 gene in ES cells with a targeting vector containing the p53.S389A mutation (*), a pGK-neo selectable marker cassette flanked by loxP sites, and a pGK-DTA selectable marker cassette for negative selection. Homologous integration of the vector results in an additional EcoRI site (in the pGK-neo promoter), which is used for identification by Southern blot analysis of neo-resistant ES cell clones using probe I. Deletion of the pGK-neo marker cassette was accomplished by crossing mice with the p53.S389A mutation with Deleter mice expressing Cre in their germ line and was verified by Southern blot analysis using a BamHI digest and probe II. The resulting mutant p53.S389A allele differs from the wild-type allele, in addition to having the desired p53.S389A mutation in p53 exon 11, only in the presence of one loxP site downstream of the p53 gene. Expression of the p53.S389A mutation was verified by RT-PCR, followed by sequencing of the entire p53 coding sequence using RNA isolated from several tissues of p53.S389A mice. (B, BamHI; R, EcoRI; H, HindIII; Xho, XhoI; X, XbaI). (B) Presence of the S389A mutation was determined by PCR amplification of exon 11, followed by digestion with HinfI. The S389A mutation results in the loss of one HinfI restriction site. −, negative control.

Phosphorylation of p53.S389 and total p53 protein levels in wild-type and p53.S389A MEFs after exposure to UV or gamma radiation. Phosphorylation of Ser389 and induction of p53 protein levels in wild-type (+/+) and p53^S389A/S389A (SA/SA) MEFs after treatment with 20 J of UV-C/m² (A) or 5 Gy of gamma radiation (B). Protein extracts were prepared from MEFs at different time points after the treatment, and phosphorylation of Ser389 and total p53 protein levels were visualized by Western blotting after immunoprecipitation. The remnant was moved to an additional Western blot, which was incubated with actin antibody to control for loading.

DNA binding by p53 protein after UV irradiation. EMSAs were performed using a p53-specific oligonucleotide probe, in wild-type (+/+), p53^S389A/S389A (SA/SA), and p53^−/− (−/−) MEFs. All samples were isolated 6 h after irradiation with 20-J/m² UV-C and were incubated with the labeled probe in the presence or absence of p53-specific antibody and wild-type (WT) cold competitor.

Expression of p53 target genes in MEFs. Wild-type (•), p53^S389A/S389A (□), and p53^−/− (×) MEFs were exposed to 20-J/m² UV-C radiation. At 6 and 16 h after treatment, RNA was isolated and RT-PCR was performed on different p53 target genes. The relative gene expression was normalized to the gene expression of GAPDH in the same sample, and average values with standard deviations from triplicate reactions are shown. Note that a different graduation scale is applied to each graph.

UV-induced apoptosis and gamma radiation-induced cell cycle arrest in MEFs. (A) Wild-type (+/+), p53^S389A/+ (SA/+), p53^S389A/S389A (SA/SA), and p53^−/− (−/−) MEFs were exposed to 12- or 20-J/m² UV-C radiation, and the apoptotic response was measured 16 h later with a cell death ELISA detection assay. The apoptotic response in UV-irradiated samples is normalized to the apoptotic response in untreated cultures from the same MEF cell line. The apoptotic response of each genotype is subsequently depicted relative to the response in wild-type cells. For each data point, at least three independent MEF cell lines of each genotype were analyzed in independent experiments. (B) Wild-type (○), p53^S389A/+ (▵), p53^S389A/S389A (□), and p53^−/− (×) MEFs were exposed to 20-J/m² UV-C radiation, and the apoptotic response was measured 8, 16, 24, 32, and 36 h later with a trypan blue exclusion apoptosis assay. The apoptotic response in UV-irradiated MEFs is normalized similarly to that in Fig. . For each data point, at least two independent MEF cell lines of each genotype were analyzed in independent experiments. (C) Wild-type (+/+), p53^S389A/+ (SA/+), p53^S389A/S389A (SA/SA), and p53^−/− (−/−) MEFs were exposed to 5 Gy of gamma radiation, and cell cycle arrest was determined 18 h later by FACS analysis. Left, representative examples of FACS analysis. Right, quantitation of arrest responses of MEFs to gamma radiation. The degree of G₁ arrest is indicated by a ratio of the S-phase fraction of cells after gamma exposure to the S-phase fraction of untreated cells. Each bar represents the average and standard deviation of multiple experiments. FL1-H, BrdU incorporation; FL2-A, DNA content.

In vivo and in vitro apoptosis in thymocytes from p53.S389A mice. (A) For analysis of in vivo-induced apoptosis, mice of the indicated genotypes (+/+, wild type; SA/+, p53^S389A/+; SA/SA, p53^S389A/S389A; and −/−, p53^−/−) were exposed to a single dose of gamma radiation (5 Gy; whole body). Twenty-four and 48 h later, thymocytes were isolated, and the percentage of CD4/CD8-double-positive cells was determined by FACS analysis. The bars represent three independent experiments (i.e., mice). Open bars, untreated thymocytes; speckled bars, 24 h after gamma irradiation; hatched bars, 48 h after gamma irradiation. (B) For analysis of in vitro-induced apoptosis, thymocytes were isolated from mice of different genotypes and propagated in cell culture. The cells were either left untreated or exposed to increasing doses (as indicated) of doxorubicin (p53-dependent apoptosis) or dexamethasone (p53-independent apoptosis). After 24 h, the thymocytes were stained with PI and annexin V, and the viable cells were counted by FACS analysis. The relative percentage of viable cells (negative for both PI and annexin V) for each sample is shown. All values are normalized to the number of cells remaining viable in untreated cultures derived from the same animal and stained simultaneously. The data represent at least two independent experiments (i.e., mice). ○, wild-type; ▵, p53^S389A/+; □, p53^S389A/S389A; ×, p53^−/−.

Survival of p53.S389A mice. Mice of the indicated genotypes were monitored as they aged for development of tumors or other aberrations to determine survival. Moribund animals were killed, and their tissues were analyzed by histopathology. Genotypes: wild-type, ○; p53^S389A/+, ▵; p53^S389A/S389A, □; p53^+/−, no symbol. The number of animals analyzed per genotype is indicated in Table . Each group consisted of approximately equal numbers of males and females. wks, weeks.

UV-B-induced skin tumor development in p53.S389A mice. (A) Mice were bred into a hairless background (SKH:HR7) and exposed to a daily UV-B radiation dose of 600 J/m². Wild-type, ○; p53^S389A/+, ▵; p53^S389A/S389A, □; p53^+/−, no symbol; wks, weeks. (B) Representative examples of squamous cell carcinoma and squamous cell papilloma, both isolated from UV-B-irradiated p53^S389A/S389A mice.