PMC

Doxorubicin-induced apoptosis in ES cells. Wild-type D3 (▴), p53^+/− (●), p53^−/− (■), p53^+/R270H (○), and p53^+/P275S (□) ES cells were grown to subconfluency, and exposed to either 1 or 2 μg/ml doxorubicin in the culture medium. After 24 hr, the cells were stained with PI and annexin V antibody, and the numbers of viable cells were determined by fluorescence-activated cell sorter analysis. Data are averages of four independent experiments.

Generation of p53 point mutated alleles in ES cells. Scheme for targeting one allele of the murine p53 gene in ES cells with a targeting vector containing either the R270H or the P275S mutation (asterisk), and a neo-TK selectable marker cassette flanked by LoxP sites (first selection round). Homologous integration of the vector results in an additional EcoRI site (situated in the promoter of pMC-neo), which is used for Southern blot analysis of neomycin-resistant ES cell clones. In the homologous recombinant clones excision of the neo-TK selectable marker cassette was accomplished by transfection with circular pMC-CreN plasmid (second selection round). The resulting allele differs from the wild-type allele, besides the R270H or P275S mutation, only in the presence of one LoxP site downstream of the coding sequences.

Expression levels of p53 target genes in ES cells upon treatment with γ irradiation. (A) Wild-type D3 (+/+); p53^+/− (+/−); p53^−/− (−/−); p53^+/R270H (+/R270H); and p53^+/P275S (+/P275S) ES cells were grown for 2 days, and exposed to a single dose of γ irradiation (500 cGy). At 1, 3, and 6 hr after the treatment, RNA was isolated. Northern blots were probed with bax, cyclinG, mdm2, and gapdh cDNA probes. The 0-hr time points represent untreated cells, isolated at the same time as the 3-hr time point after γ irradiation. (B) Quantitation of Northern blot signals normalized for expression levels of gapdh (used as a loading control). Note that in the bax graph, the scale of the y axis is different from the other axes. ▴, Wild-type D3; ●, p53^+/−; ■, p53^−/−; ○, p53^+/R270H; □, p53^+/P275S.

The effect of heterozygous point mutations in p53 on apoptosis in thymocytes. (A) p53-dependent and -independent apoptosis. Thymocytes of mice of all different genotypes were isolated (▴, wild type; ●, p53^+/−; ■, p53^−/−; ○, p53^+/R270H; □, p53^+/P275S) and exposed in vitro to γ irradiation (500 cGy), doxorubicin (0.2 μg/ml), or dexamethasone (1 μM). At the time points indicated, thymocytes were stained with annexin V and PI. 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 stained simultaneously. Data are representatives of ≥2 independent experiments (i.e., mice). (B) Northern blot of p53 target genes in thymocytes after γ irradiation (500 cGy). Two or 5 hr after the treatment, RNA was isolated, and Northern blots were probed with bax, cyclinG, and gapdh cDNA probes. (C) Quantitation of Northern blot signals normalized for expression levels of gapdh (used as a loading control). ▴, Wild type; ●, p53^+/−; ■, p53^−/−; ○, p53^+/R27OH; and □, p53^+/P275S.