XPA-deficient Mice Research Articles

The distinct roles of NEIL1 and XPA in limiting aflatoxin B₁-induced mutagenesis in mice.

Dietary exposure to aflatoxin B₁ (AFB₁) is a risk factor for the development of hepatocellular carcinomas (HCCs). Following metabolic activation, AFB₁ reacts with guanines to form covalent DNA adducts, which induce high-frequency G > T transversions. The molecular signature associated with these mutational events aligns with the single base substitution signature 24 (SBS24) in the Catalog of Somatic Mutations in Cancer (COSMIC) database. Deficiencies in either base excision repair (BER) due to the absence of Nei-like DNA glycosylase 1 (NEIL1) or nucleotide excision repair (NER) due to the absence of xeroderma complementation group A protein (XPA) contribute to HCCs in murine models. In the current study, ultra-low error duplex sequencing was used to characterize mutational profiles in liver DNAs of NEIL1-deficient, XPA-deficient, and DNA repair-proficient mice following neonatal injection of 1 mg/kg AFB₁. Analyses of AFB₁-induced mutations showed high cosine similarity to SBS24, regardless of repair proficiency status. The absence of NEIL1 resulted in an approximately 30% increase in the frequency of mutations, with distribution suggesting preferential NEIL1-dependent repair of AFB₁ lesions in open chromatin regions. A trend of increased mutagenesis was also observed in the absence of XPA. Consistent with the role of XPA in transcription-coupled repair, mutational profiles in XPA-deficient mice showed disruption of the transcriptional bias in mutations associated with SBS24. Implications: Our findings define the roles of DNA repair pathways in AFB₁-induced mutagenesis and carcinogenesis in murine models, with these findings having implications in human health for those with BER and NER deficiencies.

Potential UV-Protective Effect of Freestanding Biodegradable Nanosheet-Based Sunscreen Preparations in XPA-Deficient Mice.

Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disorder. As patients with XP are deficient in nucleotide excision repair, they show severe photosensitivity symptoms. Although skin protection from ultraviolet (UV) radiation is essential to improve the life expectancy of such patients, the optimal protective effect is not achieved even with sunscreen application, owing to the low usability of the preparations. Nanosheets are two-dimensional nanostructures with a thickness in the nanometer range. The extremely large aspect ratios of the nanosheets result in high transparency, flexibility, and adhesiveness. Moreover, their high moisture permeability enables their application to any area of the skin for a long time. We fabricated preparations containing avobenzone (BMDBM) based on freestanding poly (L-lactic acid) (PLLA) nanosheets through a spin-coating process. Although monolayered PLLA nanosheets did not contain enough BMDBM to protect against UV radiation, the layered nanosheets, consisting of five discrete BMDBM nanosheets, showed high UV absorbance without lowering the adhesive strength against skin. Inflammatory reactions in XPA-deficient mice after UV radiation were completely suppressed by the application of BMDBM-layered nanosheets to the skin. Thus, the BMDBM layered nanosheet could serve as a potential sunscreen preparation to improve the quality of life of patients with XP.

1114 Voriconazole enhances skin tumor development by UVB through the mechanism its induction of inflammatory response

We previously shown that inflammatory response induced by ultraviolet B (UVB)/reactive oxygen species/DNA damage has a pivotal role for skin tumor development. It has known that some drugs as chlorothiazide induced photo-sensitivity reactions. Recently there have been reports that squamous cell carcinoma in sun exposed areas developed after administration of voriconazole, anti-fungal agent. We studied the effects of inflammatory response with or without voriconazole using xeroderma pigmentosum complementation group A model mice (Xpa-deficient mice). Skin carcinogenesis by repetitive UVB exposure with voriconazole on Xpa-deficient mice were also investigated. We compared the serum level of cytokines from Xpa-deficient mice irradiated by UVB or UVA and administered with voriconazole, chlorothiazide and vehicle control by cytokine array assay. We exposed UVB on the groups of each drug administered along with wild-type mice twice a week for 10 weeks-UVB exposure for the evaluation of skin tumor development. The serum of Xpa-deficient mice of voriconazole or chlorothiazide and UVB significantly elevated wide variety of cytokines compared with vehicle only administration. Both agents increase the expression of CXCLl1, M-CSF, CCL2, CCL12, CXCL12, TNFα, GCSF and IL16 after UVB exposure, furthermore, the serum of voriconazole plus UVB exposure up-regulated exclusively the level of IFN-γ, IL1α, IL1β, IL4, IL7, IL13 and CCL9. Chronic UVB exposure on Xpa-deficient mice with voriconazole administration significantly showed much more tumors, tumor volumes and faster induction compared with vehicle group. The mechanism of skin tumor development in sun-exposed area after administration of voriconazole indicates that voriconazole induced strong inflammatory response following DNA damage by UVB.

CXCL1 Inhibition Regulates UVB-Induced Skin Inflammation and Tumorigenesis in Xpa-Deficient Mice

Xeroderma pigmentosum complementation group A is a hereditary disease characterized by early onset of skin cancers and freckle-like pigmented maculae in sun-exposed sites. Although the etiology of the predisposition to UVR-induced skin tumors in xeroderma pigmentosum complementation group A is well investigated as a repair deficiency in UVR-induced DNA damage, the mechanism of exaggerated sunburn in patients with xeroderma pigmentosum complementation group A and whether UVR-induced inflammation relates to a skin tumor-prone phenotype remains to be elucidated. Using gene profiling of xeroderma pigmentosum complementation group A model mice, Xpa-deficient mice, we found that expression of CXCL1 in the skin and blood of Xpa-deficient mice increased significantly after UVB exposure over even a limited area compared with that of wild-type mice. We administered CXCL1 neutralizing antibody or the antioxidant agent, N-acetylcysteine, to Xpa-deficient mice after UVB irradiation and found significant suppression of blood levels of CXCL1, ear swelling and erythema, the hallmarks of inflammation and neutrophil chemotaxis. Xpa-deficient mice treated with chronic UVB exposure plus administration of CXCL1 neutralizing antibody or N-acetylcysteine yielded many fewer skin tumors compared with the control group. This indicates that the UVB-induced strong inflammatory response of Xpa-deficient mice plays a role in skin tumor development, which could be suppressed by regulating chemokines such as CXCL1.

Dietary grape seed proanthocyanidins inactivate regulatory T cells by promoting NER-dependent DNA repair in dendritic cells in UVB-exposed skin.

Ultraviolet B (UVB) radiation induces regulatory T cells (Treg cells) and depletion of these Treg cells alleviates immunosuppression and inhibits photocarcinogenesis in mice. Here, we determined the effects of dietary grape seed proanthocyanidins (GSPs) on the development and activity of UVB-induced Treg cells. C3H/HeN mice fed a GSPs (0.5%, w/w)-supplemented or control diet were exposed to UVB (150 mJ/cm2) radiation, sensitized to 2,4-dinitrofluorobenzene (DNFB) and sacrificed 5 days later. FACS analysis indicated that dietary GSPs decrease the numbers of UVB-induced Treg cells. ELISA analysis of cultured sorted Treg cells indicated that secretion of immunosuppressive cytokines (interleukin-10, TGF-β) was significantly lower in Treg cells from GSPs-fed mice. Dietary GSPs also enhanced the ability of Treg cells from wild-type mice to stimulate production of IFNγ by T cells. These effects of dietary GSPs on Treg cell function were not found in XPA-deficient mice, which are incapable of repairing UVB-induced DNA damage. Adoptive transfer experiments revealed that naïve recipients that received Treg cells from GSPs-fed UVB-irradiated wild-type donors that had been sensitized to DNFB exhibited a significantly higher contact hypersensitivity (CHS) response to DNFB than mice that received Treg cells from UVB-exposed mice fed the control diet. There was no significant difference in the CHS response between mice that received Treg cells from UVB-irradiated XPA-deficient donors fed GSPs or the control diet. Furthermore, dietary GSPs significantly inhibited UVB-induced skin tumor development in wild-type mice but not in XPA-deficient mice. These results suggest that GSPs inactivate Treg cells by promoting DNA repair in dendritic cells in UVB-exposed skin.

Hearing Dysfunction in Xpa-Deficient Mice.

Xeroderma pigmentosum (XP) is a rare recessive heredity disease caused by DNA repair impairment characterized by photosensitivity and neurologic symptoms in half of the cases. There are eight subtypes of XP: XP-A–XP-G and XP variant. Among eight subtypes, XP complementation group A (XP-A) display the lowest DNA repair ability and the severest cutaneous and neurologic symptoms. While its pathogenesis of skin symptoms have been well-studied, that of neurological symptoms, including sensorineural hearing loss (SNHL) remains unknown. Basic studies have suggested that SNHL may be caused by inner ear damage, including damage to the spiral ganglion neurons and organ of Corti, and that the XP-A is associated with most severe form of SNHL in humans. Here, we report the occurrence of SNHL in Xpa-deficient mice. Xpa-deficient mice and wild-type mice underwent measurements for auditory brainstem response, and the results revealed that Xpa-deficient mice exhibited significantly greater (p < 0.01) ABR thresholds at 4, 8, and 16 kHz than the wild-type mice. Furthermore, the number of spiral ganglion neurons was reduced in Xpa-deficient mice compared with that in wild-type mice, indicating that hearing loss may be related to spiral ganglion neuron deficiency, consistent with the few reports published in human patients with XP. These results provide important insights into the pathogenesis of SNHL in patients with XP-A.

601. Gene Therapy for Cockayne Syndrome

Cockayne Syndrome (CS) is a rare autosomal recessive genetic disorder. The majority of CS cases involve mutations in either the CSA (25%) or CSB (75%) gene, leading to defective transcription-coupled nucleotide excision repair and RNA polymerase II mediated transcription. Clinically this presents as progressive degeneration of the central nervous system, retina, cardiovascular system, and cochlea, causing mental retardation, post-natal growth defects, pigmented retinopathy, cataracts, dermal UV sensitivity, organ dysfunction and shortened life expectancy.CSA and CSB single mutant mice display retinal degeneration with age and ocular sensitivity to ionizing radiation. Because limited publications on these mouse models exist we began by characterizing the retinal dysfunction through histology and electroretinography (ERG). We found that the retinal degeneration in CSA and CSB mutant mice (C57Bl6 background) was occurring over a slower time course (over 1 year) than previously reported. Therefore we aimed to increase the rate of retinal degeneration in these animals using UV light exposure, whole body radiation, or high intensity light exposure. Our goal was to increase the speed of retinal degeneration to a timeframe that is more readily studied for the purposes of gene therapy correction. Preliminary data indicates that we can cause a decrease in the scotopic ERG (b-wave) 2 weeks after high-dose ultraviolet light exposure (100000J/m^2), a change not seen in C57Bl6 mice at the same UV dose. There was also a decrease in b-wave amplitude following radiation exposure (2-3 gray) in the CSB mouse model that was not seen in C57Bl6 mice. Histology on these mice is currently pending. Improving the rate of photoreceptor loss will allow us to perform rAAV based gene therapy to deliver the CSB gene back to the retina and ascertain whether UV and radiation resistance can be restored.While single mutant CSA and CSB mice do not have significant neurological deficits, when crossed with XPA deficient mice they develop clinical signs similar to severely affected human patients. Signs in these mice include neurologic deficits, poor weight gain and death around postnatal day 21. These mice could be ideal candidates to show that correction of both the central nervous system disease as well as the retinal degeneration is possible in a single model. To determine if both the brain and retina could be targeted in the same animal (and therefore model what may be necessary in the human patients) we have looked at targeting both organs simultaneously using either a single IV injection in neonatal mice or a combined intra-cerebral ventricular (ICV) injection and IV injection in the same animal. We found that delivery of the rAAV vector (regardless of serotype tested) into the retro-orbital sinus at postnatal day 1-3 leads to widespread transduction of the retina. Temporal vein delivery postnatal day 1-3 only resulted in transduction of the inner layer of the retina, not the photoreceptors. When ICV and IV injections were combined in neonates widespread brain and retinal transduction was achieved.These preliminary data indicate that a murine model for retinal gene therapy of CS is feasible and that potentially both the retina and brain could be targeted in a single model.

Abstract 1276: UV radiation interacts with Endothelin 3 and loss of nucleotide excision repair pathway to promote melanomagenesis.

Abstract Melanomagenesis is influenced by the interaction of environmental (such as UV radiation) and genetic factors. The Xpa gene is important in the Nucleotide Excision Repair (NER) pathway. Similar to humans afflicted with Xeroderma Pigmentosum, which results from mutations in genes that play a role in NER, Xpa deficient mice show high sensitivity to UV light, leading to skin cancer development. However, Xpa deficient mice do not develop melanoma even upon UV exposure. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. In humans, this pathway has also been implicated in melanoma progression and its metastatic potential. The purpose of this study is to develop a UV induced melanoma mouse model that combines Xpa deficiency with the over-activation of the Edn3 pathway. To this end, transgenic mice over-expressing Edn3 under the control of the keratin 5 promoter (K5-Edn3) and carrying a targeted mutation in Xpa were exposed to a single suberythemal neonatal dose of UV radiation. A subgroup of mice was also exposed to a second dose of UV radiation at 6 weeks of age. Histomorphology and immunostaining were used to confirm the melanocytic origin of primary skin tumors and metastases. Melanoma was only found in animals with the K5-Edn3 transgene. High penetrance was observed in animals exposed to one dose of UV radiation that were Xpa null (100%, n=3) when compared to Xpa heterozygous (73%, n=11) or Xpa wild type (43%, n=7). Enlarged and hyperpigmented lymph nodes were present in animals with melanoma skin lesions and these were diagnosed as local metastases. These results indicate that UV radiation exposure, in conjunction with over-activation of the Edn3 pathway is sufficient to lead to melanomagenesis in mice. Citation Format: Ana Paula Benaduce, Deannys Batista, Gabriel Grilo, Karen Jorge, Lidia Kos. UV radiation interacts with Endothelin 3 and loss of nucleotide excision repair pathway to promote melanomagenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1276. doi:10.1158/1538-7445.AM2013-1276

Silymarin inhibits ultraviolet radiation-induced immune suppression through DNA repair-dependent activation of dendritic cells and stimulation of effector T cells

Silymarin inhibits UVB-induced immunosuppression in mouse skin. To identify the molecular mechanisms underlying this effect, we used an adoptive transfer approach in which dendritic cells (DCs) from the draining lymph nodes of donor mice that had been UVB-exposed and sensitized to 2,4,-dinitrofluorobenzene (DNFB) were transferred into naïve recipient mice. The contact hypersensitivity (CHS) response of the recipient mice to DNFB was then measured. When DCs were obtained from UVB-exposed donor mice that were not treated with silymarin, the CHS response was suppressed confirming the role of DCs in the UVB-induced immunosuppression. Silymarin treatment of UVB-exposed donor mice relieved this suppression of the CHS response in the recipients. Silymarin treatment was associated with rapid repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in DCs and silymarin treatment did not prevent UV-induced immunosuppression in XPA-deficient mice which are unable to repair UV-induced DNA damage. The CHS response in mice receiving DCs from silymarin-treated UV-exposed donor mice also was associated with enhanced secretion of Th1-type cytokines and stimulation of T cells. Adoptive transfer of T cells revealed that transfer of either CD8+ or CD4+ cells from silymarin-treated, UVB-exposed donors resulted in enhancement of the CHS response. Cell culture study showed enhanced secretion of IL-2 and IFNγ by CD8+ T cells, and reduced secretion of Th2 cytokines by CD4+ T cells, obtained from silymarin-treated UVB-exposed mice. These data suggest that DNA repair-dependent functional activation of DCs, a reduction in CD4+ regulatory T-cell activity, and stimulation of CD8+ effector T cells contribute to silymarin-mediated inhibition of UVB-induced immunosuppression.

Platelet-activating Factor Receptor Agonists Mediate Xeroderma Pigmentosum A Photosensitivity

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.

Abstract 1845: Silymarin, a phytochemical from milk thistle, inhibits ultraviolet radiation-induced apoptosis by stimulating DNA repair in skin cells

Abstract Over exposure to solar ultraviolet (UV) radiation is the major etiological factor for over one million new cases of non-melanoma skin cancers in the US each year. One of the hallmark events of exposure to UVB radiation (290-320 nm) is the induction of apoptotic cell death of keratinocytes, the results of which are evident within the epidermis as sunburn cells. The formation of sunburn cells is a protective mechanism for limiting survival of cells with irreparable DNA damage. Because of its protective function, alterations in UVB-induced apoptosis may have a profound impact in the induction of skin cancer. We have shown that topical application of silymarin, a flavonoid from milk thistle (Silybum marianum L.), inhibits UVB-induced immunosuppression in mice, and this protective effect is mediated through the induction of immunoregulatory cytokine IL-12. By using in vitro cell culture and in vivo genetically-modified mouse models, here we report that UVB-induced apoptotic cell death of epidermal keratinocytes was suppressed by silymarin, and in this process UVB-induced DNA damage was significantly reduced or repaired by silymarin-induced IL-12-mediated induction of nucleotide excision repair. Using normal human epidermal keratinocytes (NHEK) as an in vitro model, we found that exposure of NHEK to UVB (20 mJ/cm2) radiation induces apoptosis which was suppressed by pretreatment of NHEK with silymarin (5-20 µg/ml) when analyzed by flow cytometry, and the levels of damaged DNA was determined by dot-blot analysis and immunocytostaining using antibody specific to thymine dimers. The suppression of UVB-induced apoptosis after silymarin treatment was related with the enhancement of IL-12 in silymarin-treated NHEK. Treatment of silymarin-treated NHEK with anti-IL-12 antibody abrogated the anti-apoptotic effect of silymarin in UVB-exposed NHEK which was confirmed by the analysis of DNA damaging cells using cytostaining and comet assay on per cell level. To further confirm the role of silymarin-induced IL-12 in prevention of UVB-induced apoptosis, we used IL-12p40 knock out (IL-12 KO) mice. We observed that UVB-induced sunburn cells were resolved more rapidly in the skin of wild-type mice treated topically with silymarin than untreated control mice. In contrast, the extent of UVB-induced numbers of sunburn cells was not significantly different in the silymarin-treated IL-12 KO mice and untreated control mice. In addition, treatment of xeroderma pigmentosum complementation group A (XPA)-deficient mice, with silymarin did not promote the repair of UVB-induced sunburn cells while promoted the repair of sunburn cells in wild-type of XPA-deficient mice, indicating that silymarin can protect keratinocytes from apoptosis induced by DNA-damaging UV radiation by inducing DNA repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1845. doi:10.1158/1538-7445.AM2011-1845

Functional and physical interactions between ERCC1 and MSH2 complexes for resistance to cis-diamminedichloroplatinum(II) in mammalian cells

Bulky DNA lesions are mainly repaired by nucleotide excision repair (NER), in which the interaction of ERCC1 with XPA protein recruits the ERCC1–XPF complex, which acts as a structure-specific endonuclease in the repair process. However, additional functions besides NER have been suggested for the ERCC1–XPF complex, because ERCC1- or XPF-deficient rodent cells are significantly more sensitive to DNA interstrand cross-linking (ICL) agents such as cis-diamminedichloroplatinum(II) (CDDP) than any other NER-deficient cells and because ERCC1-deficient mice suffer a more severe phenotype than XPA-deficient mice. By using RNA interference we show here that suppression of ERCC1 expression increases the sensitivity of xeroderma pigmentosum group A (XPA)-deficient human cells to CDDP but not to UV. This increased sensitivity to CDDP is observed in mouse cells defective in Xpa as well but not in cells defective both in Xpa and the mismatch repair gene Msh2. These data suggest that ERCC1 and MSH2 are involved co-operatively in CDDP resistance in mammalian cells. As a possible molecular basis, we show further a physical interaction between endogenous ERCC1 and MSH2 complexes in HeLa cell extracts. Using tagged ERCC1 in COS7 cells, the minimum region in ERCC1 necessary for the immuno-precipitation of MSH2 is turned out to be the carboxyl-terminal domain between the 184th and 260th amino acid, which is partly overlapping with the XPF-binding domain of ERCC1. This interaction may be important in additional functions of ERCC1–XPF including the repair of CDDP-induced DNA damage.

Enhanced spontaneous and aflatoxin-induced liver tumorigenesis in xeroderma pigmentosum group A gene-deficient mice.

Xeroderma pigmentosum (XP) is an autosomal recessive hereditary disease featuring defective nucleotide excision repair (NER). XP patients are highly sensitive to sunlight and develop skin cancer at an early age. While the fact that XP patients have a large increase in mortality from skin cancers has been extensively documented, the relation between XP and internal tumors has received little attention. We therefore analyzed development of spontaneous and aflatoxin B(1) (AFB(1))-induced liver tumors in XPA-deficient congenic mice, originally created by repeated back-crosses with inbred C3H/HeN mice. Spontaneous liver tumors were assessed at the age of 16 months in two separate experiments using F5 and F10 lines. The incidence of and average number of spontaneous tumors per mouse were significantly higher in XPA-/- than in XPA+/+ and +/- mice. Similarly, F10 XPA-/- mice receiving i.p. injection of 0.6 or 1.5 mg/kg b.w. AFB(1) at 7 days of age demonstrated more liver tumors than their heterozygous or homozygous positive counterparts when examined at month 11. These results demonstrate that XPA-deficient mice have increased susceptibility to both spontaneous liver tumor development and AFB(1)-induced hepatocarcinogenesis.

Photobiologic and photoimmunologic characteristics of XPA gene-deficient mice.

Xeroderma pigmentosum group A (XPA) gene-deficient mice cannot repair UV-induced DNA damage and easily develop skin cancers by UV irradiation. Just like human XP patients, homozygous (-/-) mice developed stronger longer-lasting acute inflammation than did wild-type mice after a single irradiation with UVB. Moreover, the model mice showed more severe UV-induced damage of keratinocytes and Langerhans cells than did the control mice. UVB-induced local and systemic immunosuppression was greatly enhanced in the (-/-) mice. Treatment with indomethacin, an inhibitor of prostaglandin (PG) synthesis, inhibited UV-induced inflammation and abrogated immunosuppression. In XPA-deficient mice, the amount of PGE2 and the expression level of COX-2 mRNA greatly increased after UVB irradiation compared with wild-type mice. These results suggest that the excess DNA photoproducts remaining in XPA-deficient cells after UV radiation induce COX-2 expression and subsequently produce a high amount of PGE2, which causes the enhancement of inflammation and immunosuppression. In XPA-deficient mice, the natural killer cell activity significantly decreased after repeated exposures to UVB. Our experimental data indicate that cancer development in XP patients involves not only mutagenesis due to the defect in DNA repair, but also the enhanced UV-immunosuppression and intensified impairment of natural killer function.

Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice

We have established xeroderma pigmentosum group A ( XPA) gene-knockout mice with nucleotide excision repair (NER) deficiency, which rapidly developed skin tumors when exposed to a low dose of chronic UV like XP-A patients, confirming that the NER process plays an important role in preventing UVB-induced skin cancer. To examine the in vivo mutation in the UVB-irradiated epidermis, we established XPA (−/−), (+/−) and (+/+) mice carrying the Escherichia coli rpsL transgene with which the mutation frequencies and spectra in the UVB-irradiated epidermal tissue can be examined conveniently. The XPA (−/−) mice showed a higher frequency of UVB-induced mutation in the rpsL transgene with a low dose (150 J/m 2) of UVB-irradiation than the XPA (+/−) and (+/+) mice, while, at a high dose (900 J/m 2) they showed almost the same frequency of mutation as the XPA (+/−) and (+/+) mice, probably because of cell death in the epidermis of the XPA (−/−) mice. However, CC→TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the XPA (−/−) mice than the XPA (+/−) and (+/+) mice at both doses of UVB. This rpsL/XPA mouse system will be useful for further analyzing the role of NER in the mutagenesis and carcinogenesis induced by various carcinogens.

Decreased UV sensitivity, mismatch repair activity and abnormal cell cycle checkpoints in skin cancer cell lines derived from UVB-irradiated XPA-deficient mice

Xeroderma pigmentosum group A gene (XPA)-deficient mice are defective in nucleotide excision repair (NER) and are therefore highly sensitive to ultraviolet (UV)-induced skin carcinogenesis. We established cell lines from skin cancers of UVB-irradiated XPA-deficient mice to investigate the phenotypic changes occurring during skin carcinogenesis. As anticipated, the skin cancer cell lines were devoid of NER activity but were less sensitive to killing by UV-irradiation than the XPA(−/−) fibroblast cell line. The lines were also more resistant to 6-thioguanine (6-TG) than XPA(−/−) and XPA(+/+) fibroblasts, which was suggestive of a mismatch repair (MMR) defect. Indeed, in vitro mismatch binding and MMR activity were impaired in several of these cell lines. Moreover, these cell lines displayed cell cycle checkpoint derangements following UV-irradiation and 6-TG exposure. The above findings suggest that MMR downregulation may help cells escape killing by UVB, as was seen previously for methylating agents and cisplatin, and thus that MMR deficient clones are selected for during the tumorigenic transformation of XPA(−/−) cells.

Delay of DNA-adduct repair and severe toxicity in xeroderma pigmentosum group A gene (XPA) deficient mice treated with 2-amino-1-methyl-6-phenyl-imidazo [4,5–b] pyridine (PhIP)

Group-A xeroderma pigmentosum (XPA) gene-deficient mice are defective in nucleotide-excision repair and highly susceptible to ultraviolet-B-, and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA)-induced skin carcinogenesis [1]. In this study, changes of 2-amino-1-methyl-6-phenylimidazo[4,5–b] pyridine (PhIP)-DNA adduct formations in the liver, colon and lung, as assessed by the 32P-postlabeling method and immunohistochemical analysis, and carcinogenic and/or toxic susceptibility of both sexes of XPA-deficient mice (XPA−/−) to PhIP, which is a carcinogenic heterocyclic amine, was examined. Levels of PhIP-DNA adduct formations in the liver, colon and lung, were almost twice as high in XPA−/− as in wild type mice (XPA+/+) mice, 7 days after a single i.g. administration of PhIP, and their delay in recovery was observed in XPA−/− mice. For the long-term experiment, XPA−/− and XPA+/+ type mice were treated with 80 ppm PhIP in the diet for the first 4 weeks followed by 40 ppm after a 2-week recovery period (long-term experiment I), or 40 ppm PhIP throughout the experiment (long-term experiment II). Severe toxicity, as evidenced by body weight retardation and poor survival, was observed in the PhIP treated XPA−/− mice of both sexes, but not in the XPA+/+. At week 40 the experiments were terminated and histopathological examinations were performed after complete autopsy. Only lymphomas/leukemias were observed as neoplastic lesions, but no significant differences were observed between the groups. As non-neoplastic lesions, degenerating changes, for example in the pancreatic acinar cells, were observed with XPA−/− mice tending to be more sensitive than XPA+/+ mice. The present study demonstrated that PhIP-DNA adduct formations in the liver, colon and lung of XPA−/− mice were demonstrated and their recovery rate was more delayed than XPA+/+ mice, and furthermore, more severe toxicity to PhIP in XPA-deficient mice was observed, but they were not susceptible to PhIP carcinogenicity under the conditions of the experiment.

Possible Involvement of Enhanced Prostaglandin E2 Production in the Photosensitivity in Xeroderma Pigmentosum Group A Model Mice

Xeroderma pigmentosum group A (XPA) gene-deficient mice cannot repair UV-induced DNA damage and easily develop skin cancers by UV irradiation. Therefore, XPA-deficient mice are a useful model of human XP and represent a promising tool for photobiologic studies of the disorder. Exposure to ultraviolet (UV) B (280-320 nm) radiation greatly enhanced inflammation and immunosuppression in these mice. To investigate the molecular mechanisms of enhanced UV inflammation and immunosuppression, we determined the amount of prostaglandin (PG) E2, an inflammatory mediator and immunomodulator, and analysed the expression of cyclooxygenase (COX) mRNA in the ear skin of XPA-deficient mice after UV irradiation. In XPA-deficient mice, the amount of PGE2 significantly increased at 48 and 72 h after UVB irradiation to the level that was 8- and 16-fold higher than those in wild-type mice, respectively. The expression level of COX-2 mRNA increased in a time-dependent manner, although COX-1 mRNA was constantly expressed. Treatment with indomethacin, a potent inhibitor of PG biosynthesis, inhibited UV-induced ear swelling, abrogated local immunosuppression, and decreased the amount of PGE2 in the ear skin of XPA-deficient mice. These results indicate that the excess DNA photoproducts remaining in XPA-deficient cells after UV radiation may induce COX-2 expression. The induced production of PGE2 may be involved in the enhanced inflammation and immunosuppression caused by UV radiation in XPA-deficient mice and XP patients.

Postnatal growth failure, short life span, and early onset of cellular senescence and subsequent immortalization in mice lacking the xeroderma pigmentosum group G gene.

The xeroderma pigmentosum group G (XP-G) gene (XPG) encodes a structure-specific DNA endonuclease that functions in nucleotide excision repair (NER). XP-G patients show various symptoms, ranging from mild cutaneous abnormalities to severe dermatological impairments. In some cases, patients exhibit growth failure and life-shortening and neurological dysfunctions, which are characteristics of Cockayne syndrome (CS). The known XPG protein function as the 3' nuclease in NER, however, cannot explain the development of CS in certain XP-G patients. To gain an insight into the functions of the XPG protein, we have generated and examined mice lacking xpg (the mouse counterpart of the human XPG gene) alleles. The xpg-deficient mice exhibited postnatal growth failure and underwent premature death. Since XPA-deficient mice, which are totally defective in NER, do not show such symptoms, our data indicate that XPG performs an additional function(s) besides its role in NER. Our in vitro studies showed that primary embryonic fibroblasts isolated from the xpg-deficient mice underwent premature senescence and exhibited the early onset of immortalization and accumulation of p53.

Accelerated accumulation of somatic mutations in mice deficient in the nucleotide excision repair gene XPA.

Inheritable mutations in nucleotide excision repair (NER) genes cause cancer-prone human disorders, such as xeroderma pigmentosum, which are also characterized by symptoms of accelerated ageing. To study the impact of NER deficiency on mutation accumulation in vivo, mutant frequencies have been determined in liver and brain of 2-16 month old NER deficient XPA-/-, lacZ hybrid mice. While mutant frequencies in liver of 2-month old XPA-/-, lacZ mice were comparable to XPA+/-, lacZ and the lacZ parental strain animals, by 4 months of age mutant frequencies in the XPA-deficient mice were significantly increased by a factor of two and increased further until the age of 16 months. In brain, mutant frequencies were not found to increase with age. These results show that a deficiency in the NER gene XPA causes an accelerated accumulation of somatic mutations in liver but not in brain. This is in keeping with a higher incidence of spontaneous liver tumors reported earlier for XPA-/- mice after about 15 months of age.