Tumor suppression by p53 in the absence of Atm

Abstract

Oncogenes can induce p53 through a signaling pathway involving p19/Arf. It was recently proposed that oncogenes can also induce DNA damage, and this can induce p53 through the Atm DNA damage pathway. To assess the relative roles of Atm, Arf, and p53 in the suppression of Ras-driven tumors, we examined susceptibility to skin carcinogenesis in 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (TPA)-treated Atm- and p53-deficient mice and compared these results to previous studies on Arf-deficient mice. Mice with epidermal-specific deletion of p53 showed increased papilloma number and progression to malignant invasive carcinomas compared with wild-type littermates. In contrast, Atm-deficient mice showed no increase in papilloma number, growth, or malignant progression. gamma-H2AX and p53 levels were increased in both Atm(+/+) and Atm(-/-) papillomas, whereas Arf(-/-) papillomas showed much lower p53 expression. Thus, although there is evidence of DNA damage, signaling through Arf seems to regulate p53 in these Ras-driven tumors. In spontaneous and radiation-induced lymphoma models, tumor latency was accelerated in Atm(-/-)p53(-/-) compound mutant mice compared with the single mutant Atm(-/-) or p53(-/-) mice, indicating cooperation between loss of Atm and loss of p53. Although p53-mediated apoptosis was impaired in irradiated Atm(-/-) lymphocytes, p53 loss was still selected for during lymphomagenesis in Atm(-/-) mice. In conclusion, in these models of oncogene- or DNA damage-induced tumors, p53 retains tumor suppressor activity in the absence of Atm.

Figure 1. Skin tumor development in Atm and p53 deficient mice

A, DMBA/TPA induced papillomas in Atm deficient mice. The mean number of papillomas per mouse of the indicated size class is plotted over time post DMBA treatment. B, The average number of papillomas of all size classes from mice of the indicated Atm genotype is plotted over time post DMBA treatment. C, Carcinoma free survival in DMBA/TPA treated mice. (p=0.8 Atm−/− vs Atm+/+ and p=0.2 for Atm +/− vs Atm +/+, log rank test). D, Tumor free survival in DMBA/TPA treated mice. (p<0.0001 for Atm−/− vs. Atm+/+ and p= 0.11 for Atm+/− vs. Atm+/+, using log rank test). E, DMBA/TPA induced papillomas in Trp53^F2-10/F2-10 (p53 WT) and Trp53^F2-10/F2-10; K14-Cre (skin-specific p53 deficient) mice. The number of papillomas in p53 deficient mice at 23 and 25 weeks was greater then wild types (p= 0.003 and 0.04 respectively, using an unpaired t-test). F, Carcinoma free survival in DMBA/TPA treated p53 WT and skin specific p53 deleted mice (p53 conditional KO). The difference was highly significant (p= 0.001 using the log rank test).

Figure 2. DNA damage signaling in Atm deficient papillomas

A, H&E stained sections and immunostaining for the indicated proteins from papillomas from wild type, Atm−/− and Arf−/− mice. Top row was photographed with a final magnification of 100x and lower rows at 600x. B, Quantification of γ-H2AX, pChk2, p53, apoptosis, (APO) and mitotic index (MI), in Atm +/+, Atm−/− and Arf−/− papillomas (n=5–7 tumors per genotype). The values were calculated as mean plus standard deviation of the number of cells that stained for γ-H2AX, pChk2, p53, or apoptotic or mitotic figures per 400x field. The p values were determined by the Mann-Whitney test.

Figure 3. Loss of p53 and Atm cooperate during tumorigenesis

A, Spontaneous tumor development is accelerated in Atm−/−p53−/− (n=13) mice relative to Atm−/− (n=14) or p53−/− (n=18) mice. B, IR-induced tumor development is accelerated in all genotypes except Atm−/− mice. Atm−/−p53−/− mice (n=4) develop tumors faster then Atm−/− (n=8) or p53−/− (n=15) mice. C, IR induced apoptosis in thymus from 3 week old mice (4 Gy 4 h). The values are mean plus standard deviation of apoptotic figures per 400x field for 3–5 mice per genotype.

Figure 4. p53 LOH is seen in tumors from both Atm+/+ and Atm−/− mice

PCR analysis shows loss of the wild type allele of p53 in 4/7 spontaneous and 8/9 IR -induced tumors from Atm−/−p53+/− mice and from 4/7 tumors from p53+/− mice. * indicates LOH of p53 in tumors. wt=wild type p53 allele, mu= mutant p53 allele.