Search

Supplementary Table S3 from Lack of p53 Ser389 Phosphorylation Predisposes Mice to Develop 2-Acetylaminofluorene–Induced Bladder Tumors but not Ionizing Radiation–Induced Lymphomas

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. [Cancer Res 2007;67(10):4648–56]

Supplementary Figure S3 from Lack of p53 Ser389 Phosphorylation Predisposes Mice to Develop 2-Acetylaminofluorene–Induced Bladder Tumors but not Ionizing Radiation–Induced Lymphomas

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

Supplementary Table 1 from Dominant-Negative but not Gain-of-Function Effects of a p53.R270H Mutation in Mouse Epithelium Tissue after DNA Damage

Genome maintenance is considered a prime longevity assurance mechanism as apparent from many progeroid human syndromes that are caused by genome maintenance defects. The ERCC1 protein is involved in three genome maintenance systems: nucleotide excision repair, interstrand cross-link repair, and homologous recombination. Here we describe in-life and post-mortem observations for a hypomorphic Ercc1 variant, Ercc1 −/Δ7, which is hemizygous for a single truncated Ercc1 allele, encoding a protein lacking the last seven amino acids. Ercc1 −/Δ7 mice were much smaller and median life span was markedly reduced compared to wild-type siblings: 20 and 118 weeks, respectively. Multiple signs and symptoms of aging were found to occur at an accelerated rate in the Ercc1 −/Δ7 mice as compared to wild-type controls, including a decline in weight of both whole body and various organs, numerous histopathological lesions, and immune parameters. Together they define a segmental progeroid phenotype of the Ercc1 −/Δ7 mouse model.

The carcinogenic potential of chemicals and pharmaceuticals is traditionally tested in the chronic, 2 year rodent bioassay. This assay is not only time consuming, expensive and often with a limited sensitivity and specificity but it also causes major distress to the experimental animals. A major improvement in carcinogenicity testing, especially regarding reduction and refinement of animal experimentation, could be the application of toxicogenomics. The ultimate aim of this study is to demonstrate a proof-of-principle for transcriptomics biomarkers in various tissues for identification of (subclasses of) carcinogenic compounds after short-term in vivo exposure studies. Both wild-type and DNA repair-deficient Xpa(-/-)/p53(+/-) (Xpa/p53) mice were exposed up to 14 days to compounds of three distinct classes: genotoxic carcinogens (GTXC), non-genotoxic carcinogens (NGTXC) and non-carcinogens. Subsequently, extensive transcriptomics analyses were performed on several tissues, and transcriptomics data were screened for potential biomarkers using advanced statistical learning techniques. For all tissues analyzed, we identified multigene gene-expression signatures that are, with a high confidence, predictive for GTXC and NGTXC exposures in both mouse genotypes. Xpa/p53 mice did not perform better in the short-term bioassay. We were able to achieve a proof-of-principle for the identification and use of transcriptomics biomarkers for GTXC or NGTXC. This supports the view that toxicogenomics with short-term in vivo exposure provides a viable tool for classifying (geno)toxic compounds.

Patients carrying mutations in the XPB helicase subunit of the basal transcription and nucleotide excision repair (NER) factor TFIIH display the combined cancer and developmental-progeroid disorder xeroderma pigmentosum/Cockayne syndrome (XPCS). Due to the dual transcription repair role of XPB and the absence of animal models, the underlying molecular mechanisms of XPB(XPCS) are largely uncharacterized. Here we show that severe alterations in Xpb cause embryonic lethality and that knock-in mice closely mimicking an XPCS patient-derived XPB mutation recapitulate the UV sensitivity typical for XP but fail to show overt CS features unless the DNA repair capacity is further challenged by crossings to the NER-deficient Xpa background. Interestingly, the Xpb(XPCS) Xpa double mutants display a remarkable interanimal variance, which points to stochastic DNA damage accumulation as an important determinant of clinical diversity in NER syndromes. Furthermore, mice carrying the Xpb(XPCS) mutation together with a point mutation in the second TFIIH helicase Xpd are healthy at birth but display neonatal lethality, indicating that transcription efficiency is sufficient to permit embryonal development even when both TFIIH helicases are crippled. The double-mutant cells exhibit sensitivity to oxidative stress, suggesting a role for endogenous DNA damage in the onset of XPB-associated CS.

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. [Cancer Res 2007;67(10):4648–56]

The nucleotide excision repair (NER) pathway comprises two sub-pathways, transcription coupled repair (TCR) and global genome repair (GGR). To establish the importance of these separate sub-pathways in tumor suppression, we exposed mice deficient for either TCR (Csb), GGR (Xpc) or both (Xpa) to 300 ppm 2-acetylaminofluorene (in feed, ad libitum) in a unique comparative exposure experiment. We found that cancer proneness was directly linked to a defect in the GGR pathway of NER as both Xpa and Xpc mice developed significantly more liver tumors upon 2-AAF exposure than wild type or Csb mice. In contrast, a defect in TCR appeared to act tumor suppressive, leading to a lower hepatocellular tumor response in Xpa mice (tumor incidence of 25%) as compared to Xpc mice (53% tumor-bearing mice). The link between deficient GGR and tumor proneness was most pronounced in the liver, but this phenomenon was also found in the urinary bladder. As tumor induction by 2-AAF appeared almost exclusively dependent on a defect in GGR, we examined whether gene mutation induction in the non-transcribed lacZ locus could reliably predict tumor risk. Interestingly, however, short-term 2-AAF exposure induced lacZ mutant levels in Csb mice almost as high as those found in Xpa or Xpc mice. This indicates that lacZ mutant frequencies are not correlated with a specific DNA repair defect and eventual tumor outcome, at least not in the experimental design presented here.

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.

Phytoestrogens, like isoflavonoids and lignans, have been postulated as possible colorectal cancer protective constituents. To investigate this hypothesis, two high-fiber sources rich in lignan precursors, i.e., rye bran and flaxseed, were tested for their ability to modulate intestinal tumor development in ApcMin mice. Test diets consisted of a control diet (a Western-style diet, adjusted for fiber and/or phytate content) supplemented with 5% flaxseed or 30% rye bran. Chemical analysis of diets and blood samples confirmed the enhanced systemic exposure of mice fed the test diets to the major lignan precursors, i.e., secoisolariciresinol and matairesinol. No statistically significant difference was observed in the incidence and multiplicity of small intestinal and colon tumors at terminal sacrifice between mice fed the control diet or the diet supplemented with 5% flaxseed. With the rye bran diet a statistically significant enhancement of the number of small intestinal tumors in female mice was observed. The number of colon tumors, however, was comparable between the control and rye bran-fed mice of either sex. Furthermore, no activating point mutations in the K-ras oncogene nor positive immunohistochemical staining for the p53 gene were observed in a set of 48 colon tumors. In conclusion, our results demonstrate that increased intake of lignan precursors from flaxseed or rye bran, administered in a Western-style diet, does not protect against intestinal tumor development in an appropriate animal model for intestinal neoplasia such as the ApcMin mice.

Combined oral benzo[a]pyrene and inhalatory ozone exposure have no effect on lung tumor development in DNA repair-deficient Xpa mice.Hoogervorst EM, de Vries A, Beems RB, van Oostrom CT, Wester PW, Vos JG, Bruins W, Roodbergen M, Cassee FR, Vijg J, van Schooten FJ, van Steeg H.National Institute of Public Health and the Environment, Laboratory of Toxicology, Center for Environment and Health Research, Bilthoven, The Netherlands.There is considerable concern about an enhanced risk of lung tumor development upon exposure of humans to polycyclic aromatic hydrocarbons (PAHs), like benzo[a] pyrene (B[a]P), in combination with induced lung cell proliferation by toxic agents like ozone. We studied this issue in wild-type (WT) C57BL/6 mice, the cancer prone nucleotide excision repair-deficient Xeroderma pigmentosum complementation group A mice (Xpa-/-) and the even more sensitive Xpa-/-/p53+/- mice. The mice were treated with B[a]P through the diet at a dose of 75 p.p.m., in combination with intermittent ozone exposures (0.8 p.p.m.). First, a dose-range finding study with WT and Xpa-/- mice was conducted to determine the optimal ozone concentration giving high cell proliferation and low toxic side effects. We show by BrdU incorporation that cell proliferation in the lung was induced by ozone, with an optimal concentration of 0.8 p.p.m., which was subsequently used in the (sub)chronic studies. In the subchronic study, in which lacZ mutant frequency and BPDE-DNA adduct formation were measured, the mice were treated for 13 weeks with B[a]P and/or ozone, whereas in the chronic study this treatment protocol was followed by a 6 month period on control feed and filtered air. As expected, oral B[a]P exposure appeared to be highly carcinogenic to Xpa-/- and Xpa-/-/p53+/- mice and to a lesser extent to WT mice. A high incidence of forestomach tumors and some tumors of the esophagus were found. In the lung, a clear genotoxic effect of B[a]P was found as shown by the presence of BPDE-DNA adducts. However, these DNA adducts in combination with induction of cell proliferation did not result in increased lacZ mutations, nor in lung tumor formation not even in the highly sensitive Xpa-/- and Xpa-/-/p53+/- mice. The implication of these findings for tumor risk assessment will be discussed.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease in which repair of ultraviolet (UV)-induced DNA damage is impaired or is totally absent due to mutations in genes controlling the DNA repair pathway known as nucleotide excision repair (NER). XP is characterized, in part, by extreme sensitivity of the skin to sunlight, and XP patients have a more than 1000-fold increased risk of developing cancer at sun-exposed areas of the skin. To study the role of NER in chemical-induced tumorigenesis in more detail, the authors developed Xpa-/- homozygous knockout mice with a complete defect in NER (designated as Xpa mice or XPA model). Xpa mice develop skin tumors at high frequency when exposed to UV light, and as such, they mimic the phenotype of human XP. Moreover, the Xpa mice also appear to be susceptible to genotoxic carcinogens given orally. Based on these phenotypic characteristics, the Xpa mice were considered to be an attractive candidate mouse model for use in identifying human carcinogens. In an attempt to further increase both the sensitivity and specifi city of the XPA model in carcinogenicity testing, the authors crossed Xpa mice with mice having a heterozygous defect in the tumor suppressor gene p53. Xpa/p53+/- double knockout mice develop tumors earlier and with higher incidences upon exposure to carcinogens as compared to their single knockout counterparts. Here the authors describe the development and features of the Xpa mouse and present some examples of the Xpa and Xpa/p53+/- mouse models' sensitivity towards genotoxic carcinogens. It appeared that the Xpa/p53 +/- double knockout mouse model is favorable over both the Xpa and p53+/- single knockout models in short-term carcinogenicity testing. In addition to the fact that the double knockout mice respond more robustly to carcinogens, they also appear to respond in a very discriminative way. All compounds identified thus far are true (human) carcinogens, and, therefore, the authors believe that the Xpa/p53+/- mouse model is an excellent candidate for a future replacement of the chronic mouse bioassay, at least for certain classes of chemicals.

XPA-deficient mice have a complete deficiency in nucleotide excision repair, and as such they display a cancer predisposition after exposure to several carcinogens. Besides being sensitive to genotoxic agents applied to the skin, they are also susceptible to human carcinogens given orally, like benzo[a]pyrene (B[a]P). To study the role of the tumor suppressor gene p53 in DNA repair, gene mutation, and tumor induction, we crossed XPA-deficient mice with p53 knockout mice and lacZ (pUR288) gene marker mice. When treated orally (by gavage) with B[a]P, the XPA−/−/p53+/− double transgenic mice developed tumors much earlier and with higher frequency compared to their single transgenic counterparts. The major tumor type found in all genotypes was generalized lymphoma mainly residing in the spleen; several sarcomas were observed in p53+/− and XPA−/−/p53+/− mice. Next, we determined lacZ mutation frequencies in several (non)target tissues. It appeared that in the spleen (the major tumor target tissue) of XPA−/− and XPA−/−/p53+/− mice the lacZ mutation frequency was significantly elevated (80–100 × 10−5), and was two times higher as found in spleens of B[a]P-treated WT and p53+/− mice (P = 0.003). In nontumor target tissues like liver and lung, we found a moderate increase in the lacZ gene mutation frequency (30–40 × 10−5), which was independent of the genotype. The results obtained with the DNA-repair deficient XPA mice indicate that a significantly increased lacZ mutation frequency in a particular organ/tissue is an early marker for tumor development at later stages at the same site. However, the synergistic effect of a XPA−/−- and a p53+/−-deficiency in tumor development is not reflected by an absolute increase in the lacZ mutation frequency in the major tumor target tissue of XPA−/−/p53+/− or p53+/− mice compared to that of XPA−/− and WT mice, respectively. Environ. Mol. Mutagen. 34:124–130, 1999 © 1999 Wiley-Liss, Inc.

Defects in the xeroderma pigmentosum complementation group A-correcting (XPA) gene, which encodes a component of the nucleotide excision repair (NER) pathway, are associated with the cancer-prone human disease xeroderma pigmentosum. We previously generated mice lacking the XPA gene, which develop normally but are highly sensitive to ultraviolet-B and 7,12-dimethylbenz[a] anthracene-induced skin tumors. Here we report that XPA-deficient mice spontaneously developed hepatocellular adenomas at a low frequency as they aged. Furthermore, oral treatment of XPA-deficient mice with the carcinogen benzo[a]pyrene (B[a]P) resulted in the induction of mainly lymphomas. These tumors appeared earlier and with a higher incidence than in B[a]P-treated wild-type and heterozygous mice. Our results show for the first time that XPA-deficient mice also displayed an increased sensitivity to developing tumors other than tumors of the skin.

The metabolism of iron and copper in male Nagase analbuminaemic (NA) and Sprague Dawley (SD) rats was compared. Relative liver weight was higher and spleen weight significantly lower in NA than SD rats. In NA rats, red blood cell count, haemoglobin and haematocrit were lower, whereas plasma transferrin, total iron-binding capacity and mean corpuscular haemoglobin were higher when compared with SD rats. Iron concentrations in plasma, liver, kidneys and heart were higher, and those in the spleen and tibia were lower, in NA rats. The iron concentrations in liver and spleen were positively correlated with the amount of brown pigment as observed histopathologically. Bile flow as well as biliary iron and copper excretion were higher in NA than SD rats. Copper concentrations in liver, kidneys and plasma were higher in NA rats. Plasma levels of ceruloplasmin were about two-fold higher in NA rats. The feeding of a high-iron diet reduced kidney copper concentrations in both strains of rats, which was associated with a decrease in the absorption and biliary excretion of copper.

This study was designed to compare the effects of a calcium antagonist (isradipine) and a converting enzyme inhibitor (ramipril) on progression and regression of atherosclerosis in hypercholesterolemic rabbits. Sixty rabbits in three groups were fed a 0.3% cholesterol diet for 4 weeks. After this induction period, group II received the 0.3% cholesterol diet, group III received cholesterol diet with isradipine (0.33 mg/kg/day), and group IV received cholesterol with ramipril (0.33 mg/kg/day) for 12 more weeks. A group of 20 rabbits received a standard diet throughout the study (group I). After 16 weeks, 10 rabbits were randomly chosen from each group and used in the progression study. The other rabbits were placed on a standard diet and remained on their respective drug regimen for 12 more weeks. In the progression phase of the study, ramipril significantly attenuated the percentage of aortic lesions in group IV (35 +/- 6%) as compared with group II (56 +/- 6%, p < 0.05), whereas isradipine had no effect. Acetylcholine (ACh)-induced maximum endothelium-dependent relaxations (EDR) of aortic rings were significantly reduced by the atherogenic diet to 37 +/- 4 versus 77 +/- 2% in group I (p < 0.05). Treatment with ramipril significantly improved maximum EDR to 53 +/- 3% (p < 0.05 vs. group II). Isradipine had no significant effect on impaired EDR. Aortic rings with endothelium from group II developed supersensitivity to sodium nitroprusside (SNP) and had significantly reduced basal cyclic GMP levels as compared with those of group I. Both drugs prevented development of supersensitivity to SNP and blunted the cholesterol-induced reduction in basal cyclic GMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

We report the effects of isradipine and ramipril on regression of diet-induced atherosclerosis in rabbits. Regression of diet-induced atherosclerosis was not significantly affected by ramipril, but isradipine significantly retarded regression. Thirty rabbits in three groups were fed a 0.3% cholesterol diet for 4 weeks. After this induction period, group IIr received the 0.3% cholesterol diet, group IIIr received the 0.3% cholesterol diet with isradipine (0.33 mg/kg/day), and group IVr received the 0.3% cholesterol diet with ramipril (0.33 mg/kg/day) for 12 more weeks. The rabbits then received a standard diet and remained on their respective drug regimen for 12 more weeks. Group Ir (10 rabbits) received a standard diet for 28 weeks. Acetylcholine (ACh)-induced maximal endothelium-dependent relaxations (EDR) of aortic rings were significantly less in group IIr (22.8 +/- 3.2%) than in group Ir (66.4 +/- 4.0%; p < 0.05). Ramipril and isradipine did not improve EDR as compared with group IIr. Regression of atherosclerosis was accompanied by an improved endothelium-dependent releasing factor (EDRF) release from the endothelium, but ramipril and isradipine did not promote this process. In addition, regression was associated with increasing sensitivity of vascular smooth muscle to EDRF that was significantly retarded by isradipine but not ramipril. Basal cyclic GMP levels were significantly reduced in aortic rings from group IIr as compared with group Ir. Ramipril, but not isradipine, restored basal cyclic GMP levels to control values. Both isradipine and ramipril protect against endothelial degeneration in hypercholesterolemic rabbits. However, isradipine but not ramipril inhibits regression of diet-induced atherosclerosis in rabbits.

In this study we examined the effect of 4,12 and 16 weeks of hypercholesterolemia, induced by feeding a 0.3% cholesterol enriched diet, on endothelial function in rabbit aorta. Endothelial function was assessed by measuring acetylcholine (ACh) induced endothelium dependent relaxations in isolated aorta and ACh induced release of Endothelium Derived Relaxing Factor (EDRF) from the endothelium. In all groups (4, 12 and 16 weeks) hypercholesterolemia was accompanied by a significant rise of total serum cholesterol and HDL-cholesterol/total cholesterol ratio. The intirnal surface of the total aorta covered by atherosclerotic fatty streaks after 4, 12 and 16 weeks of hypercholesterolemia was 33.8 ± 3.3%, 44.1 ± 5.2% and 55.6 ± 6.1% respectively. After 4 weeks of hypercholesterolemia endothelium dependent relaxations to ACh were augmented while ACh induced EDRF release was not affected. In contrast 12 and 16 weeks of hypercholesterolemia significantly reduced both ACh induced EDRF release and endothelium depend...

The DNA repair-deficient Xpa(-/-)p53(+/-) (Xpa/p53) mouse is a potent model for carcinogenicity testing, representing increased sensitivity toward genotoxic but surprisingly also toward true human non-genotoxic carcinogens. The mechanism of this increased sensitivity in Xpa/p53 mice toward non-genotoxic carcinogens is still unknown. Here, we investigated the mechanism of the human non-genotoxic carcinogen cyclosporine A (CsA) in the Xpa/p53 mouse model. Xpa/p53 mice exposed to CsA for 39 weeks showed a significantly increased lymphoma incidence as compared with untreated Xpa/p53 mice and CsA-treated wild-type (WT) mice. We excluded concealed genotoxicity of CsA in Xpa/p53 mice by mutant frequency analyses. As a next step, we used a genetic approach: immunodeficient DNA-PKcs mice, defective in the catalytic subunit of the DNA-dependent protein kinase, were crossed with Xpa and Xpa/p53 mice. Xpa/p53 mice had an increased lymphoma incidence with shorter latency times as compared with DNA-PKcs-deficient WT and Xpa mice. Surprisingly, also six of 15 DNA-PKcs/Xpa/p53 females had developed an adenocarcinoma of the mammary gland. Tumor responses in CsA-treated and DNA-PKcs-deficient Xpa/p53 mice were comparable as both genotypes developed mainly splenic lymphomas enriched in B lymphocytes. From our present studies, we hypothesize that levels of initiated precancerous cells are elevated in Xpa/p53 mice. These cells are insufficiently eliminated due to either suppression of the immune system by CsA or through immune-related DNA-PKcs deficiency. Based on the current studies and those conducted previously, we conclude that the Xpa/p53 model is an excellent adjunct to the current chronic rodent bioassay.

The accumulation of DNA damage is a slow but hazardous phenomenon that may lead to cell death, accelerated aging, and cancer. One of the most versatile defense mechanisms against the accumulation of DNA damage is nucleotide excision repair, in which, among others, the Xeroderma pigmentosum group C (XPC) and group A (XPA) proteins are involved. To elucidate differences in the functions of these two proteins, comprehensive survival studies with Xpa−/−, Xpc−/− and wild-type control female mice in a pure C57BL/6J background were done. The median survival of Xpc−/− mice showed a significant decrease, whereas the median survival of Xpa−/− mice did not. Strikingly, Xpa−/− and Xpc−/− mice also showed a phenotypical difference in terms of tumor spectrum. Xpc−/− mice displayed a significant increase in lung tumors and a trend toward increased liver tumors compared with Xpa-deficient or wild-type mice. Xpa−/− mice showed a significant elevation in liver tumors. Additionally, Xpc-deficient mice exhibited a strong increase in mutant frequency in lung compared with Xpa−/− mice, whereas in both models mutant frequency is increased in liver. Our in vitro data displayed an elevated sensitivity to oxygen in Xpc−/− in mouse embryonic fibroblasts (MEF) when compared with Xpa−/− and wild-type fibroblasts. We believe that XPC plays a role in the removal of oxidative DNA damage and that, therefore, Xpc−/− mice display a significant increase in lung tumors and a significant elevation in mutant frequency in lung, and Xpc-deficient MEFs show greater sensitivity to oxygen when compared with Xpa−/− and wild-type mice. [Cancer Res 2008;68(5):1347–53]

Background Systematic, high-throughput studies of mouse phenotypes have been hampered by the inability to analyze individual animal data from a multitude of sources in an integrated manner. Studies generally make comparisons at the level of genotype or treatment thereby excluding associations that may be subtle or involve compound phenotypes. Additionally, the lack of integrated, standardized ontologies and methodologies for data exchange has inhibited scientific collaboration and discovery. Results Here we introduce a Mouse Phenotype Analysis System (MPHASYS), a platform for integrating data generated by studies of mouse models of human biology and disease such as aging and cancer. This computational platform is designed to provide a standardized methodology for working with animal data; a framework for data entry, analysis and sharing; and ontologies and methodologies for ensuring accurate data capture. We describe the tools that currently comprise MPHASYS, primarily ones related to mouse pathology, and outline its use in a study of individual animal-specific patterns of multiple pathology in mice harboring a specific germline mutation in the DNA repair and transcription-specific gene Xpd. Conclusion MPHASYS is a system for analyzing multiple data types from individual animals. It provides a framework for developing data analysis applications, and tools for collecting and distributing high-quality data. The software is platform independent and freely available under an open-source license 1.

Breast cancer is a major public health problem among women worldwide. Phytoestrogens and dietary fat composition are being investigated to elucidate the role of nutrition in breast cancer risk. Both epidemiological and rodent studies suggest that the chemopreventive effect of phytoestrogens depends on timing of exposure. We investigated spontaneous mammary tumor development in female heterozygous MMTV/c-neu (Tg.NK) mice upon isoflavone exposure on background diets rich in either n-6 or n-3 polyunsaturated fatty acids (PUFAs). Three different exposure protocols were used, either from conception to weaning, or from weaning onwards, or lifelong. Mice fed diets high in n-3 PUFAs developed mammary tumors 15 weeks later than mice fed n-6 PUFA diets. In the latter mice, isoflavone exposure from weaning onwards resulted in a significant decrease in tumor incidence and a delay in tumor onset. Therefore, the effects of phytoestrogen exposure on tumor formation appear to depend on the composition of the background diet and on the timing of exposure within the life cycle.

The tumor suppressor gene p53 has an apparent role in breast tumor development in humans, as ∼30% 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 p53R270H/+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 p53R270H/+WAPCre mice is estrogen receptor α positive, and expression profiles of genes also implicated in human breast cancer appear similarly altered. As such, p53R270H/+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.

Chronic use of phenacetin-containing analgesics has been associated with the development of renal cancer. To establish genotoxicity as a possible cause for the carcinogenic effect of phenacetin, we exposed wild type and DNA repair deficient Xpa-/- and Xpa-/-/Trp53+/- mice (further referred as Xpa and Xpa/p53 mice, respectively), carrying a reporter lacZ gene, to 0.75% (w/w) phenacetin mixed in feed. Xpa mice completely lack the nucleotide excision repair pathway, and as such they are sensitive to some classes of genotoxic compounds. Phenacetin exposure induced a significant increase of lacZ mutations in the kidney of both Xpa and Xpa/p53 mice. A minor response was found in liver, whereas no lacZ mutation induction was observed in the spleen of these animals. Interestingly, the observed phenacetin-induced mutant frequencies were higher in male than those found in female mice. This gender difference is probably due to a difference in metabolic rate. Phenacetin-induced mutations mainly consisted of point mutations rather than deletions. The mutational spectra in the kidney of treated WT and Xpa mice were quite similar. Taken together, these results demonstrate that the human carcinogen phenacetin acts as a weak genotoxic agent in an in vivo mouse model system.

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.

As part of the international evaluation program coordinated by ILSI/HESI, the potential of DNA repair deficient Xpa- /- mice and the double knockout Xpa- /- .p53+ /- mice for short term carcinogenicity assays was evaluated. For comparison also wild-type C57BL/6 mice (WT) were included in these studies. Four test compounds were administered to groups of 15 male and 15 female Xpa- /- mice, Xpa- /- .p53+ /- mice and WT mice for 39 weeks. The model compounds investigated were haloperidol, reserpine (nongenotoxic rodent carcinogens, putative human noncarcinogens), phenacetin (genotoxic rodent carcinogen, suspected human carcinogen), and D-mannitol (noncarcinogen in rodents and humans). The test compounds were administered as admixture to rodent diet at levels up to 25 mg/kg diet for haloperidol, 7.5 mg/kg diet for reserpine, 0.75% for phenacetin, and 10% for D-mannitol. These levels included the maximum tolerable dose (MTD). Survival was not affected with any of the test compounds. Haloperidol, reserpine and D-mannitol were negative in the carcinogenicity assay with Xpa- /- and Xpa- /- .p53+ /- mice, showing low and comparable tumor incidences in controls and high-dose animals. The results obtained with phenacetin may be designated equivocal in Xpa- /- .p53+ /- mice, based on the occurrence of a single rare tumor in the target organ (kidney) accompanied by a low incidence of hyperplastic renal lesions and a high incidence of karyomegaly. These results are in agreement with the currently known carcinogenic potential of the 4 test compounds in humans.

Xeroderma pigmentosum (XP) patients are deficient in nucleotide excision repair (NER) because of mutations in one of the genes coding for NER enzymes. This results predominantly in high frequency of UV-induced skin tumors at an early age; the most severe phenotype is found in patients of complementation group A (XPA). However, in a subset of these XPA patients no skin tumors appear, even at advanced age. Fibroblasts of this subset of patients are not capable of raising UV-induced enhanced reactivation (ER) of viruses and up-regulation of ornithine decarboxylase (ODC). We hypothesized that prevention of ODC induction would protect NER-deficient patients from cancer. To simulate the situation in XPA patients, we used a hairless Xpa knockout mouse model and down-regulated the ODC activity by difluoromethylornithine (DFMO) administered in the drinking water. The DFMO treatment significantly suppressed UV-induced carcinogenesis. In a crossover study, we additionally found that discontinuation of the DFMO treatment resulted in a rapid appearance of skin tumors, up to levels found in mice not treated with DFMO. Late-stage DFMO treatment significantly reduced the number of carcinomas by a factor of 2-3, and it appeared to select for carcinomas with high ODC activity. These results indicate that DFMO suppresses the outgrowth but not the initiation of UV-induced tumors. The DFMO treatment reduced the tumor load but did not offer the Xpa knockout mice full protection against UV carcinogenesis.

Thymomas are usually found in the anterior thorax as encapsulated, smoothly surfaced or coarsely lobulated tumors, consisting of firm, gray-white to pink-white tissue which does not adhere to adjacent tissue. Their diameter can be up to 3 cm. Smaller tumors are generally found incidentally. They are seen as distinct nodules or as local thickening in the thymus, which disturb the symmetrical appearance of the organ. Larger tumors are more likely to cause clinical signs and be a possible cause of death due to compression of intrathoracic organs. Rarely, gross infiltration of the tumor into the surrounding tissues is seen. Careful collection and examination of residual normal thymus tissue is often helpful in differential diagnosis.

The thymus consists of two poorly encapsulated lobes which arise in the embryo as separate primordia on each side of the midline in the neck region. During ontogeny, the organ migrates caudally and medially to the superior mediastinum where the two lobes become closely connected, although they do not appear to fuse. The relative weight is largest about a week after birth; the absolute weight is largest at about the age of 2 months and then gradually declines. In old rats, especially in preterminal condition, the organ can be so small that it is hardly recognizable in the mediastinal fat tissue.

Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is caused by a defect in the transcription-coupled repair subpathway of nucleotide excision repair (NER). Here, complete inactivation of NER in Csbm/m/Xpa−/− mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csbm/m/Xpa−/− mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis, and die before weaning. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the growth hormone/insulin-like growth factor 1 (GH/IGF1) somatotroph axis and oxidative metabolism, increased antioxidant responses, and hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csbm/m/Xpa−/− and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural ageing and the DNA repair–deficient mice. Importantly, wild-type mice exposed to a low dose of chronic genotoxic stress recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis., Studies in mice defective in two DNA repair pathways (global NER and TCR; an animal model for Cockayne syndrome) highlight a link between aging, a failure to repair DNA lesions, and metabolic alterations., Author Summary Normal metabolism routinely produces reactive oxygen species that damage DNA and other cellular components and is thought to contribute to the ageing process. Although DNA damage is typically kept in check by a variety of enzymes, several premature ageing disorders result from failure to remove damage from active genes. Patients with Cockayne syndrome (CS), a genetic mutation affecting one class of DNA repair enzymes, display severe growth retardation, neurological symptoms, and signs of premature ageing followed by an early death. Whereas mouse models for CS exhibit relatively mild deficits, we show that concomitant inactivation of a second DNA repair gene elicits severe CS pathology and ageing. Moreover, a few days after birth, these mice undergo systemic suppression of genes controlling growth, an unexpected decrease in oxidative metabolism, and an increased antioxidant response. Similar physiological changes are also triggered in normal mice by chronic exposure to DNA-damaging oxidative stress. From these findings, we conclude that DNA damage triggers a response aimed at limiting oxidative DNA damage levels (and associated tissue degeneration) to extend lifespan and promote healthy ageing. Better understanding of the ageing process will help to delineate intervention strategies to combat age-associated pathology.

The modifying effect of beta-carotene on benzo[a]pyrene (BP)-induced tumors of the respiratory tract was investigated in Syrian hamsters. Groups of hamsters were fed a semisynthetic diet supplemented with either no or 56 mg/kg beta-carotene. Respiratory tract tumors were induced by intratracheal instillation of BP attached to ferric oxide. The beta-carotene and vitamin A contents of the liver were increased in the high beta-carotene group, but the serum beta-carotene levels were very low when compared with those commonly observed in humans. beta-Carotene supplementation did not affect the tumor response of the respiratory tract. Neither the incidence and severity of preneoplastic changes were influenced. However, there was a statistically significant inverse relationship between serum retinol content and the presence of respiratory tract tumors in survivors, regardless of the dietary treatment.