Esther M. Hoogervorst

Increased Sensitivity to UV Radiation in Mice with a p53 Point Mutation at Ser389

ABSTRACT Phosphorylation is important for p53 protein stabilization and activation after DNA damage. Serine 389 of p53 is specifically phosphorylated after UV irradiation, whereas gamma radiation activates p53 through a different pathway. To study the in vivo significance of p53 phosphorylation at serine 389, we generated a physiological mouse model in which p53 phosphorylation at serine 389 is abolished by alanine substitution. Homozygous mutant p53.S389A mice are viable and have an apparently normal phenotype. However, cells isolated from these mice are partly compromised in transcriptional activation of p53 target genes and apoptosis after UV irradiation, whereas gamma radiation-induced responses are not affected. Moreover, p53.S389A mice show increased sensitivity to UV-induced skin tumor development, signifying the importance of serine 389 phosphorylation for the tumor-suppressive function of p53.

Dominant-Negative but not Gain-of-Function Effects of a p53.R270H Mutation in Mouse Epithelium Tissue after DNA Damage

p53 alterations in human tumors often involve missense mutations that may confer dominant-negative or gain-of-function properties. Dominant-negative effects result in inactivation of wild-type p53 protein in heterozygous mutant cells and as such in a p53 null phenotype. Gain-of-function effects can directly promote tumor development or metastasis through antiapoptotic mechanisms or transcriptional activation of (onco)genes. Here, we show, using conditional mouse technology, that epithelium-specific heterozygous expression of mutant p53 (i.e., the p53.R270H mutation that is equivalent to the human hotspot R273H) results in an increased incidence of spontaneous and UVB-induced skin tumors. Expression of p53.R270H exerted dominant-negative effects on latency, multiplicity, and progression status of UVB-induced but not spontaneous tumors. Surprisingly, gain-of-function properties of p53.R270H were not detected in skin epithelium. Apparently, dominant-negative and gain-of-function effects of mutant p53 are highly tissue specific and become most manifest upon stabilization of p53 after DNA damage.

Lack of p53 Ser389 Phosphorylation Predisposes Mice to Develop 2-Acetylaminofluorene–Induced Bladder Tumors but not Ionizing Radiation–Induced Lymphomas

Cellular activity of the tumor suppressor protein p53 is primarily regulated by posttranslational modifications. Phosphorylation of the COOH terminus, including Ser389, is thought to result in a conformational change of the p53 protein, enhancing DNA binding and transcriptional activity. In vitro studies presented here show that, in addition to UV radiation, Ser389 is phosphorylated upon exposure to 2-acetylaminofluorene (2-AAF). Both agents induce bulky DNA adducts repaired by nucleotide excision repair (NER). In contrast, ionizing radiation, known to induce DNA damage not repaired by NER, does not result in Ser389 phosphorylation. Previously, we have shown that p53.S389A mutant mice, lacking the Ser389 phosphorylation site, are sensitive to developing UV-induced skin tumors. Here, we show that p53.S389A mice are also prone to developing 2-AAF-induced urinary bladder tumors, whereas no increased tumor response was found upon ionizing irradiation. These results provide evidence for our hypothesis that phosphorylation of Ser389 is important for activation of p53 to exert its function as a tumor suppressor not exclusively upon the presence of UV-induced DNA damage, but also upon exposure to other bulky adduct-inducing agents. Analysis of 2-AAF- and UV-induced tumors from p53.S389A mice revealed the presence of additional p53 mutations, indicating that lack of Ser389 phosphorylation by itself is not sufficient to abrogate p53 function in tumor suppression. In addition, analyses of skin tumors of p53.S389A mice revealed an interesting hotspot mutation previously found exclusively in NER-deficient mice and patients.

p53 heterozygosity results in an increased 2-acetylaminofluorene-induced urinary bladder but not liver tumor response in DNA repair-deficient Xpa mice.

Both nucleotide excision repair (NER) and the p53 tumor suppressor protein play crucial roles in the prevention of cells becoming cancerous. This is clearly demonstrated by the fact that NER-deficient xeroderma pigmentosum patients and Li-Fraumeni patients who carry a germ-line p53 mutation are highly tumor prone. The NER-deficient Xpa and the p53+/− mouse models clearly mimic their human counterparts, because they are both tumor prone as well. The aim of the study presented here was to analyze the relative contribution of these two pathways in tumor suppression and to analyze a possible link between NER and p53 activation in vivo. For this, we exposed Xpa, p53+/−, and Xpa/p53+/− mice to 2-acetylaminofluorene (2-AAF). We show that 2-AAF-induced urinary bladder tumor suppression is dependent on p53 status, because p53+/− mice were highly tumor prone. Xpa/p53+/− mice were even more tumor prone, whereas no increased tumor response was found in Xpa mice. Short-term assays revealed a decreased apoptotic response in Xpa/p53+/− mice, pointing in vivo toward a link between NER and p53-mediated apoptosis. In contrast, liver tumor response was primarily dependent on appropriate DNA repair, because Xpa-deficient mice were liver tumor prone. p53 heterozygosity had no influence on liver tumor incidences, in line with the results obtained from the short-term 2-AAF studies revealing no altered cellular response in p53+/− or Xpa/p53+/− mice. Interestingly, however, mice completely deficient in both NER and p53 (Xpa/p53−/− mice) showed a dramatic increase of hepatocellular proliferation accompanied by lacZ reporter gene mutations.

Genotoxic exposure: novel cause of selection for a functional ΔN-p53 isoform

The p53 gene is frequently mutated in cancers and it is vital for cell cycle control, homeostasis and carcinogenesis. We describe a novel p53 mutational spectrum, different to those generally observed in human and murine tumors. Our study shows a high prevalence of nonsense mutations in the p53 N terminus of 2-acetylaminofluorene (2-AAF)-induced urinary bladder tumors. These nonsense mutations forced downstream translation initiation at codon 41 of Trp53, resulting in the aberrant expression of the p53 isoform ΔN-p53 (or p44). We propose a novel mechanism for the origination and the selection for this isoform. We show that chemical exposure can act as a novel cause of selection for this truncated protein. In addition, our data suggest that the occurrence of ΔN-p53 accounts, at least in mice, for a cancer phenotype. We also show that gene expression profiles of embryonic stem (ES) cells carrying the ΔN-p53 isoform in a p53-null background are divergent from p53 knockout ES cells, and therefore postulate that ΔN-p53 itself has functional transcriptional properties.