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.
mice were fed for 10 days with PL (300 mg/kg) or vehicle then UV-irradiated, once. By 24 hours, UVinduced Cox-2 levels were increased in vehicle-fed and PL-fed mice, whereas by 48 and 72 hours, Cox-2 levels were four-to fivefold lower in PL-fed mice (P < 0.05). p53 expression/activity was increased in PL-fed versus vehicle-fed then UV-irradiated mice. UV-induced inflammation was decreased in PL-fed mice, as shown by ϳ60% decrease (P < 0.001) in neutrophil infiltration at 24 hours, and macrophages by ϳ50% (<0.02) at 24 and 48 hours. By 72 hours, 54 ؎ 5% cyclobutane pyrimidine dimers remained in vehiclefed versus 31 ؎ 5% in PL-fed skin (P < 0.003). The number of 8-hydroxy-2-deoxyguanosine-positive cells were decreased before UV irradiation by ϳ36% (P < 0.01), suggesting that PL reduces constitutive oxidative DNA damage. By 6 and 24 hours, the number of 8-hydroxy-2-deoxyguanosine-positive cells were ϳ59% (P < 0.01) and ϳ79% (P < 0.03) lower in PL-fed versus vehicle-fed mice. Finally, UV-induced mutations in PL-fed-mice were decreased by ϳ25% when assessed 2 weeks after the single UV exposure. These data demonstrate that PL extract supplementation affords the following photoprotective effects: p53 activation and reduction of acute inflammation via Cox-2 enzyme inhibition, increased cyclobutane pyrimidine dimer removal, and reduction of oxidative DNA damage.
The tumor suppressor gene p53 has an apparent role in breast tumor development in humans, as f30% of sporadic tumors acquire p53 mutations and Li-Fraumeni syndrome patients carrying germ line p53 mutations frequently develop breast tumors at early age. In the present study, conditional expression of a targeted mutation is used to analyze the role of the human R273H tumor-associated hotspot mutation in p53 in mammary gland tumorigenesis. Heterozygous p53 R270H/+ WAPCre mice (with mammary gland-specific expression of the p53.R270H mutation, equivalent to human R273H, at physiologic levels) develop mammary tumors at high frequency, indicating that the R270H mutation predisposes for mammary gland tumor development and acts in a dominant-negative manner in early stages of tumorigenesis. Spontaneous tumor development in these mice is further accelerated by 7,12-dimethylbenz(a)anthracene (DMBA) treatment at young age. The majority of spontaneous and DMBA-induced carcinomas and sarcomas from p53 R270H/+ WAPCre mice is estrogen receptor A positive, and expression profiles of genes also implicated in human breast cancer appear similarly altered. As such, p53 R270H/+ WAPCre mice provide a well-suited model system to study the role of p53 in breast tumorigenesis and the responsiveness of mammary gland tumors to chemotherapeutics. (Cancer Res 2005; 65(18): 8166-73)
We investigated the potential of four well-characterized amorphous silica nanoparticles to induce chromosomal aberrations and gene mutations using two in vitro genotoxicity assays. Transmission electron microscopy (TEM) was used to verify the manufacturer's nominal size of 10, 30, 80 and 400 nm which showed actual sizes of 11, 34, 34 and 248 nm, respectively. The 80 (34) nm silica nanoparticles induced chromosomal aberrations in the micronucleus assay using 3T3-L1 mouse fibroblasts and the 30 (34) and 80 (34) nm silica nanoparticles induced gene mutations in mouse embryonic fibroblasts carrying the lacZ reporter gene. TEM imaging demonstrated that the majority of nanoparticles were localized in vacuoles and not in the nucleus of 3T3-L1 cells, indicating that the observed DNA damage was most likely a result of indirect mechanisms. Further studies are needed to reveal these mechanisms and to determine the biological relevance of the effects of these particular silica nanoparticles in vivo.
Abstractp53 alterations in human tumors often involve missense mutations that may confer dominant-negative or gain-offunction 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 UVBinduced but not spontaneous tumors. Surprisingly, gainof-function properties of p53.R270H were not detected in skin epithelium. Apparently, dominant-negative and gain-offunction effects of mutant p53 are highly tissue specific and become most manifest upon stabilization of p53 after DNA damage. [Cancer Res 2007;67(10):4648-56]
The EU-EuroMix project adopted the strategy of the European Food Safety Authority (EFSA) for cumulative risk assessment, which limits the number of chemicals to consider in a mixture to those that induce a specific toxicological phenotype. These so-called cumulative assessment groups (CAGs) are refined at several levels, including the target organ and specific phenotype. Here, we explore the zebrafish embryo as a test model for quantitative evaluation in one such CAG, skeletal malformations, through exposure to test compounds 0–120 hpf and alcian blue cartilage staining at 120 hpf, focusing on the head skeleton. Reference compounds cyproconazole, flusilazole, metam, and thiram induced distinctive phenotypes in the head skeleton between the triazoles and dithiocarbamates. Of many evaluated parameters, the Meckel’s–palatoquadrate (M–PQ) angle was selected for further assessment, based on the best combination of a small confidence interval, an intermediate maximal effect size and a gentle slope of the dose–response curve with cyproconazole and metam. Additional test compounds included in the CAG skeletal malformations database were tested for M–PQ effects, and this set was supplemented with compounds associated with craniofacial malformations or cleft palate to accommodate otherwise organized databases. This additional set included hexaconazole, all-trans-retinoic acid, AM580, CD3254, maneb, pyrimethanil, imidacloprid, pirimiphos-methyl, 2,4-dinitrophenol, 5-fluorouracil, 17alpha-ethynylestradiol (EE2), ethanol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PCB 126, methylmercury, boric acid, and MEHP. Most of these compounds produced a dose–response for M–PQ effects. Application of the assay in mixture testing was provided by combined exposure to cyproconazole and TCDD through the isobole method, supporting that in this case the combined effect can be modeled through concentration addition.
XPC is one of the key DNA damage recognition proteins in the global genome repair route of the nucleotide excision repair (NER) pathway. Previously, we demonstrated that NER-deficient mouse models Xpa−/− and Xpc−/− exhibit a divergent spontaneous tumor spectrum and proposed that XPC might be functionally involved in the defense against oxidative DNA damage. Others have mechanistically dissected several functionalities of XPC to oxidative DNA damage sensitivity using in vitro studies. XPC has been linked to regulation of base excision repair (BER) activity, redox homeostasis and recruitment of ATM and ATR to damage sites, thereby possibly regulating cell cycle checkpoints and apoptosis. XPC has additionally been implicated in recognition of bulky (e.g. cyclopurines) and non-bulky DNA damage (8-oxodG). However, the ultimate contribution of the XPC functionality in vivo in the oxidative DNA damage response and subsequent mutagenesis process remains unclear. Our study indicates that Xpc−/− mice, in contrary to Xpa−/− and wild type mice, have an increased mutational load upon induction of oxidative stress and that mutations arise in a slowly accumulative fashion. The effect of non-functional XPC in vivo upon oxidative stress exposure appears to have implications in mutagenesis, which can contribute to the carcinogenesis process. The levels and rate of mutagenesis upon oxidative stress correlate with previous findings that lung tumors in Xpc−/− mice overall arise late in the lifespan and that the incidence of internal tumors in XP-C patients is relatively low in comparison to skin cancer incidence.
Phosphorylation is important in p53-mediated DNA damage responses. After UV irradiation, p53 is phosphorylated specifically at murine residue Ser389. Phosphorylation mutant p53.S389A cells and mice show reduced apoptosis and compromised tumor suppression after UV irradiation. We investigated the underlying cellular processes by time-series analysis of UV-induced gene expression responses in wild-type, p53.S389A, and p53؊/؊ mouse embryonic fibroblasts. The absence of p53.S389 phosphorylation already causes small endogenous gene expression changes for 2,253, mostly p53-dependent, genes. These genes showed basal gene expression levels intermediate to the wild type and p53 ؊/؊ , possibly to readjust the p53 network. Overall, the p53.S389A mutation lifts p53-dependent gene repression to a level similar to that of p53 ؊/؊ but has lesser effect on p53-dependently induced genes. In the wild type, the response of 6,058 genes to UV irradiation was strictly biphasic. The early stress response, from 0 to 3 h, results in the activation of processes to prevent the accumulation of DNA damage in cells, whereas the late response, from 12 to 24 h, relates more to reentering the cell cycle. Although the p53.S389A UV gene response was only subtly changed, many cellular processes were significantly affected. The early response was affected the most, and many cellular processes were phasespecifically lost, gained, or altered, e.g., induction of apoptosis, cell division, and DNA repair, respectively. Altogether, p53.S389 phosphorylation seems essential for many p53 target genes and p53-dependent processes.
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