Characterization of defective nucleotide excision repair in XPC mutant mice

doi:10.1016/S0027-5107(97)00046-8

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volume 374, Issue 1, 4 March 1997, Pages 1-9

https://doi.org/10.1016/S0027-5107(97)00046-8 Get rights and content

Abstract

Nucleotide excision repair (NER) is a fundamental process required for maintaining the integrity of the genome in cells exposed to environmental DNA damage. Humans defective in NER suffer from the hereditary cancer-prone disease xeroderma pigmentosum. In order to model this disease in mice a mutation in the mouse XPC gene was generated and used to replace a wild-type XPC allele in mouse embryonic stem cells by homologous recombination. These cells were used to derive XPC mutant mice. Fibroblasts from mutant embryos were more sensitive to the cytotoxic effects of ultraviolet light than wild-type and heterozygous cells. Repair synthesis of DNA following irradiation with ultraviolet light was reduced in these cells, indicating a defect in NER. Additionally, XPC mutant embryo fibroblasts were specifically defective in the removal of pyrimidine (6-4) pyrimidone photoproducts from the non-transcribed strand of the transcriptionally active p53 gene. Mice defective in the XPC gene appear to be an excellent model for studying the role of NER and its interaction with other proteins in the molecular pathogenesis of cancer in mammals following exposure to environmental carcinogens.

Introduction

Nucleotide excision repair (NER) is critical for maintaining the integrity of the genome following insult from various DNA-damaging agents [1]. The significance of NER to human health has been extensively documented by the study of patients with the hereditary cancer-prone disease xeroderma pigmentosum (XP) [2]. At an early age, XP patients have a dramatically increased risk of cancer in sun-exposed areas of the skin, tongue and eyes 2, 3, 4. In addition some XP individuals suffer various types of neurological deficiencies [4]. This autosomal recessive disorder has been directly associated with a defect in NER [1]. As a consequence of this defect cells from XP patients are hypersensitive to the cytotoxic effects of ultraviolet (UV) light and to UV-mimetic chemicals [5]. In addition, XP cells are hypermutable following exposure to DNA-damaging agents 6, 7. Cell fusion studies have identified 8 genetic complementation groups designated XP-A through XP-G, and XP-V (for the variant form of the disease) [2]. The most common form of XP in North America is that associated with genetic complementation group C [2]. Measurements of gene-specific and strand-specific NER have shown that XP-C cells are deficient in the repair of the non-transcribed strand of actively-transcribed genes and of transcriptionally-silent regions of the genome, but retain proficiency for NER of the transcribed strand of actively transcribed genes [8]. This is in contrast to cells of other XP complementation groups (e.g., XP-A) which are deficient in repair of both transcriptionally active and inactive regions of the genome [1].

The molecular basis of the neurological deficits in a disease characterized by defective NER is not understood. Additionally, while it might be anticipated that individuals who are constitutionally defective in NER would be at increased risk for cancers other than those associated with sunlight exposure, clinical information on the incidence of cancer of internal organs such as the respiratory and intestinal tracts, which are normally exposed to environmental carcinogens, is anecdotal for XP [3]. To examine the consequences of defective NER in the context of a mammalian organism we have generated mice that are homozygously defective in a gene required for this process. We show here that like a different XPC mutant mouse strain recently developed [9], primary mouse embryo fibroblast (MEF) cells from our strain are abnormally sensitive to killing by UV radiation. In XPC mutant MEF cells we provide direct evidence for a defect in NER. We show that like human XP-C cells, the NER defect in XPC mutant MEF cells is restricted to the non-transcribed strand of the actively transcribed p53 gene.

Section snippets

Generation of XPC mutant mice

A targeting vector was constructed that replaced a 1.2 kb XhoI fragment containing exon 10 and a portion of each of the flanking introns with a POLII-NEO selectable marker. The replacement vector containing 2.5 kb of 5′- and 6 kb of 3′-flanking homology and a diphtheria toxin A gene cassette (for negative selection) was used to transfect embryonic stem cells by electroporation. Correctly targeted embryonic stem cell clones were identified by extensive Southern blot analysis. Male germline

Generation of XPC mutant mice

A targeting vector was constructed to replace a 1.2 kb XhoI fragment of the mouse genomic XPC gene containing exon 10 and a portion of each of the flanking introns with a POLII-NEO selectable marker (Fig. 1A). This vector was used to target disruption of the XPC locus by homologous recombination in mouse embryonic stem cells (Fig. 1A,B). Correctly targeted embryonic stem cell clones were identified by Southern analysis and injected into C57Bl/6 blastocysts. Three clones produced chimeric

Discussion

The human skin cancer-prone hereditary disease XP is genetically and phenotypically complex 1, 2. Mutations in any of at least 8 distinct genetic loci have been implicated in the disease [2]. The XPA, XPB, XPC, XPD, XPF and XPG genes have been cloned and are definitively associated with NER of damaged DNA [1]. Mice that are defective in the homologous XPA gene closely mimic the cellular and clinical phenotypes of the human XP-A state, including hypersensitivity to UV light, defective NER and a

Acknowledgements

We thank our laboratory colleagues, in particular J-F Houle, for extensive discussions and for critical review of the manuscript. These studies were supported by grant CA44247 from the USPHS to ECF and EV5V-CT9100030 from the European Commission to LFHM. DLC was supported by a postdoctoral fellowship from the American Cancer Society and LBM was supported by a postdoctoral fellowship from Friends of the Center for Human Nutrition, University of Texas Southwestern Medical Center.

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Inflammation response, oxidative stress and DNA damage caused by urban air pollution exposure increase in the lack of DNA repair XPC protein
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Air pollution represents a considerable threat to health worldwide. The São Paulo Metropolitan area, in Brazil, has a unique composition of atmospheric pollutants with a population of nearly 20 million people and 9 million passenger cars. It is long known that exposure to particulate matter less than 2.5 µm (PM_2.5) can cause various health effects such as DNA damage. One of the most versatile defense mechanisms against the accumulation of DNA damage is the nucleotide excision repair (NER), which includes XPC protein. However, the mechanisms by which NER protects against adverse health effects related to air pollution are largely unknown. We hypothesized that reduction of XPC activity may contribute to inflammation response, oxidative stress and DNA damage after PM_2.5 exposure. To address these important questions, XPC knockout and wild type mice were exposed to PM_2.5 using the Harvard Ambient Particle concentrator. Results from one-single exposure have shown a significant increase in the levels of anti-ICAM, IL-1β, and TNF-α in the polluted group when compared to the filtered air group. Continued chronic PM_2.5 exposure increased levels of carbonylated proteins, especially in the lung of XPC mice, probably as a consequence of oxidative stress. As a response to DNA damage, XPC mice lungs exhibit increased γ-H2AX, followed by severe atypical hyperplasia. Emissions from vehicles are composed of hazardous substances, with polycyclic aromatic hydrocarbons (PAHs) and metals being most frequently cited as the major contributors to negative health impacts. This analysis showed that benzo[b]fluoranthene, 2-nitrofluorene and 9,10-anthraquinone were the most abundant PAHs and derivatives. Taken together, these findings demonstrate the participation of XPC protein, and NER pathway, in the protection of mice against the carcinogenic potential of air pollution. This implicates that DNA is damaged directly (forming adducts) or indirectly (Reactive Oxygen Species) by the various compounds detected in urban PM_2.5.
Loss of Epidermal HIF-1α Blocks UVB-Induced Tumorigenesis by Affecting DNA Repair Capacity and Oxidative Stress
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HIF-1α is constitutively expressed in mouse and human epidermis. It plays a crucial role in skin physiology, including the response of keratinocytes to UVR. However, little information is available about its role in photocarcinogenesis. Using a multistage model of UVB radiation-induced skin cancer, we show that the knockout of Hif-1α in the epidermis prevents tumorigenesis but at the same time triggers the formation of hyperkeratotic plaques. Our results indicate that the absence of oncogenic transformation in Hif-1α–ablated mice is related to increased DNA repair in keratinocytes, whereas the formation of hyperkeratotic plaques is caused by an increase in the levels of reactive oxygen species. Indeed, impairing the DNA repair machinery by ablating xeroderma pigmentosum C restored the UVB-induced neoplastic transformation of Hif-1α–ablated keratinocytes, whereas the development of hyperkeratotic plaques was blocked by chronic antioxidant treatment. We conclude that HIF-1α plays a procarcinogenic role in UVB-induced tumorigenesis.
NADPH Oxidase-1 Plays a Key Role in Keratinocyte Responses to UV Radiation and UVB-Induced Skin Carcinogenesis
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Citation Excerpt :
Female SKH-1 hairless mice were purchased at 4–6 weeks of age from Charles River (L’arbresle, France). Hairless Xpc mutant mice (a generous gift of N. de Wind, Leiden University Medical Center, Leiden, The Netherlands) were generated (Cheo et al., 1997) and bred into a SKH-1 hairless background as described previously (van Oosten et al., 2000). Mice were bred and maintained in a pathogen-free mouse facility at Bordeaux University.
The nicotinamide adenine dinucleotide phosphate oxidase (NOX) family enzymes are involved in several physiological functions. However, their roles in keratinocyte responses to UV radiation have not been clearly elucidated. This study shows that, among other NOX family members, UVB irradiation results in a biphasic activation of NOX1 that plays a critical role in defining keratinocyte fate through the modulation of the DNA damage response network. Indeed, suppression of both bursts of UVB-induced NOX1 activation by using a specific peptide inhibitor of NOX1 (InhNOX1) is associated with increased nucleotide excision repair efficiency and reduction of apoptosis, which is finally translated into decreased photocarcinogenesis. On the contrary, when only the second peak of UVB-induced NOX1 activation is blocked, both nucleotide excision repair efficiency and apoptosis are decreased. Our results show that inhibition of NOX1 activation could be a promising target for the prevention and treatment of UVB-induced skin cancer in nucleotide excision repair-proficient and -deficient patients.
Base Excision Repair and Nucleotide Excision Repair
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Base excision repair (BER) and nucleotide excision repair (NER) are two DNA-repair pathways that, like other DNA-repair pathways except for direct reversal of DNA damage, involve sequential activities of multiple repair proteins. The two DNA-repair pathways have been studied for more than four decades, and this investigation has elucidated their crucial roles in maintaining genomic integrity particularly in response to exposure to environmental genotoxicants including ultraviolet light, cigarette smoke carcinogens, and reactive oxygen species. This chapter introduces the history and the concepts of DNA excision repair, discusses types of DNA damage processed by BER and NER, and describes the repair mechanisms. The detailed steps in these repair pathways in cells are complex and they require the coordination of the enzymatic reactions. Recent evidence indicates that DNA-damage and excision-repair proteins are key signal transducers and play critical roles for DNA-damage responses and communications between nuclei and mitochondria. Precise knowledge of BER and NER reactions should help us understand the roles that these key DNA-repair pathways play in preserving normal physiology which in turn should improve understanding of how defects in these pathways contribute to human disease with a particular emphasis on cancer.
Genetic Evidence for XPC-KRAS Interactions During Lung Cancer Development
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The tumors were mostly adenocarcinomas in 2-month-old Xpc−/−KrasLA1 mice (Fig. 2D) whereas the same aged mice with KrasLA1 had mostly adenomas (Fig. 2C). For Xpc−/− mice, we did not observe any tumors even after one year, which is consistent with a previous report that Xpc−/− mice only develop spontaneous lung tumors at an old age (>15 months) after exposure to carcinogens (Cheo et al., 1997, 2000; Hollander et al., 2005). These data suggest that unrepaired oncogenic gene mutations resulting from the defective DNA repair system in Xpc−/− mice may be responsible for tumor development.
Lung cancer causes more deaths than breast, colorectal and prostate cancers combined. Despite major advances in targeted therapy in a subset of lung adenocarcinomas, the overall 5-year survival rate for lung cancer worldwide has not significantly changed for the last few decades. DNA repair deficiency is known to contribute to lung cancer development. In fact, human polymorphisms in DNA repair genes such as xeroderma pigmentosum group C (XPC) are highly associated with lung cancer incidence. However, the direct genetic evidence for the role of XPC for lung cancer development is still lacking. Mutations of the Kirsten rat sarcoma viral oncogene homolog (Kras) or its downstream effector genes occur in almost all lung cancer cells, and there are a number of mouse models for lung cancer with these mutations. Using activated Kras, Kras^LA1, as a driver for lung cancer development in mice, we showed for the first time that mice with Kras^LA1 and Xpc knockout had worst outcomes in lung cancer development, and this phenotype was associated with accumulated DNA damage. Using cultured cells, we demonstrated that induced expression of oncogenic KRAS^G12V led to increased levels of reactive oxygen species (ROS) as well as DNA damage, and both can be suppressed by anti-oxidants. Our results suggest that XPC may help repair DNA damage caused by KRAS-mediated production of ROS.

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Accelerated publicationCharacterization of defective nucleotide excision repair in XPC mutant mice

Abstract

Introduction

Section snippets

Generation of XPC mutant mice

Generation of XPC mutant mice

Discussion

Acknowledgements

Accelerated publication
Characterization of defective nucleotide excision repair in XPC mutant mice