DNA repair mechanisms counteract the formation of deleterious DNA lesions and maintain genomic integrity. Nucleotide excision repair (NER) is an important DNA repair pathway because of its extraordinarily large substrate specificity. P53 protein regulates NER pathway in a transcription-dependent or transcription-independent manner. Inherited polymorphisms of NER pathway genes (XPC, HR23B, XPA, DDB2, XPB, XPD, ERCC1, XPF, and XPG) and TP53 gene may contribute to individual variations in genetic susceptibility to OSCC and correlate with the prognosis of 204 OSCC patients. We carried out a hospital-based case-control study to investigate the association of 25 various polymorphisms of nine NER pathway genes and TP53 gene with the risk for OSCC. There were 34 newly diagnosed OSCC patients and 135 frequency-matched controls without BQ chewing and smoking habit as well as 313 newly diagnosed OSCC patients with BQ chewing or smoking habit and 312 frequency-matched controls being recruited between November 2003 and July 2007 at Kaoshiung Veterans General Hospital. Genotyping was performed using the PCR-RFLP techniques or TaqMan real-time PCR method. The significant association between polymorphisms of NER pathway genes and OSCC risk was mainly found among subjects with BQ chewing or smoking habit. In the single locus analysis, GA and AA genotypes of ERCC1 G-641A (AOR, 0.64; 95% CI, 0.45-0.93 and AOR, 0.48; 95% CI, 0.29-0.79, respectively; p for trend, 0.002), CT genotype of XPF C-850T (AOR, 1.53; 95% CI, 1.08-2.18; p for trend, 0.014), as well as GG genotype of XPB A-1039G (AOR, 0.51; 95% CI, 0.26-0.98; p for trend 0.034) were significantly associated with the risk of OSCC. Furthermore, -641G/ -425T or -641G/ -425C haplotype of ERCC1 (AORs, 1.34; 95% CI, 1.02-1.77 and AOR, 1.56; 95% CI, 1.18-2.07, respectively; p for trend 0.002) as well as -850T/ -247T and -850T/ -247C haplotype of XPF (AOR, 1.45; 95% CI, 1.09-1.94 for; p for trend 0.016) were strongly associated with the risk of OSCC. A trend toward increased risk of OSCC was observed when people with the increasing number of at risk genotypes in the combined analyses of nine NER pathway genes with (p for trend, <0.001) or without (p for trend 0.001) TP53 gene. Finally, in the stratification analysis, the combined effects of nine NER pathway genes had a significantly increased risk of OSCC among younger group (¡Ø50 years old), Fukienece population, BQ chewers, light smokers, or light drinkers. Besides, in the prognosis analysis of 204 OSCC patients, HR23B A-823C, polymorphisms of XPA gene, XPD C-643G, XPG C787G, and the number of at risk genotypes of NER pathway genes were associated with pathologic stage, T classification, or N classification. The association between NER genetic polymorphisms and survival of patients was only found in XPA C-1778T polymorphism. These results suggested that the single polymorphism of XPB A-1039G, ERCC1 G-641A and XPF C-850T, the joint effect of genetic polymorphisms of NER pathway genes, and gene-environment combined effect were associated with the risk of OSCC. Furthermore, in the analysis of NER genetic polymorphisms and prognosis of OSCC, we found polymorphisms of XPA gene might be a prognostic factor for OSCC.

In response to DNA damage, mammalian cells activate various DNA repair pathways to remove DNA lesions and, meanwhile, halt cell cycle progressions to allow sufficient time for repair. The nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint activation are two major cellular responses to DNA damage induced by UV irradiation. However, how these two processes are coordinated in the response is poorly understood. Here we showed that the essential NER factor XPA (xeroderma pigmentosum group A) underwent nuclear accumulation upon UV irradiation, and strikingly, such an event occurred in an ATR (Ataxia-Telangiectasia mutated and RAD3-related)-dependent manner. Either treatment of cells with ATR kinase inhibitors or transfection of cells with small interfering RNA targeting ATR compromised the UV-induced XPA nuclear translocation. Consistently, the ATR-deficient cells displayed no substantial XPA nuclear translocation while the translocation remained intact in ATM (Ataxia-Telangiectasia mutated)-deficient cells in response to UV irradiation. Moreover, we found that ATR is required for the UV-induced nuclear focus formation of XPA. Taken together, our results suggested that the ATR checkpoint pathway may modulate NER activity through the regulation of XPA redistribution in human cells upon UV irradiation.

No description available.

No description available.

No description available.

No description available.

The nucleotide excision repair pathway (NER) has been shown to efficiently remove bulky base lesions from the DNA, including those induced by solar light. It has been suggested that the NER pathway may be involved also in removing smaller oxidative base lesions from the DNA. Oxidative damage in the cell is caused by cellular aerobic respiration, with base damage to the nucleotides of the DNA being the most biologically relevant. One of the most common oxidative base lesions in the genome is the 7 ,8-dihydro-8-oxoguanine (8-oxoG). This lesion is pre-mutagenic since it can base pair with equal efficiency to the correct cytosine base, or the incorrect adenine base during DNA replication. Oxidative damage, including 8-oxoG, is repaired primarily by the base excision repair (BER) pathway, which is a multi-step, multi-protein pathway similar to NER. One key protein involved in both BER and NER is the p53 protein, which can act as a transcription factor and protein regulator to influence DNA repair. We have used a recombinant non-replicating human adenovirus, Ad5HCMVlacZ, which expresses the ~-galactosidase (~-gal) reporter gene, to examine the role of several NER proteins and the p53 protein in the BER of oxidative damage in human cells. Methylene blue (MB) acts as a photosenstizer, and after irradiation by visible light (VL) produces reactive oxygen species that cause 8-0xoG in the DNA. By infecting several normal, NER deficient and p53 deficient -tumor, primary and transformed fibroblast cell lines with a MB+VL-treated Ad5HCMVlacZ reporter construct, we were able to determine the host cell reactivation (HCR) of the oxidatively damaged reporter. Results indicate that the HCR of the MB+VL-treated reporter and the expression of p53 are enhanced by UVC pretreatment in normal human fibroblasts, suggesting that p53 may be involved in inducible BER. In addition, increased expression of p53 facilitated by pre-infection of normal cells with p53 expressing Ad5p53wt similarly enhanced HCR in the normal fibroblasts, giving further evidence that increased expression of p53 alone enhances BER. In contrast, although UVC pretreatment of p53 compromised cells resulted in enhanced HCR, the enhanced HCR did not correlate with enhanced p53 expression, suggesting that enhancement in BER can result from both p53 dependent and p53 independent mechanisms. We report also that HCR of the MB+VL-treated reporter gene was substantially reduced in SV40-transformed XP-C cells, with little or no reduction in SV40-transformed XPA, XPD, XPF, XPG and CSB cells, suggesting a role for the XPC protein in the BER ofMB+VL-induced DNA damage. In particular, the XPC protein appears to be involved in both the constitutive and inducible aspects of BER, as the HCR of the MB + VL-treated reporter was reduced in 3 UVC pretreated as well as untreated XP-C primary human fibroblast strains. In addition, pre-infection of cells with Ad5p53wt, resulted in an enhanced HCR of normal but not XP-C deficient fibroblasts consistent with a p53 dependent involvement of the XPC protein in BER of MB+VL-treated DNA. Additional studies were also conducted to determine the cell sensitivity of normal and NER deficient SV40-transformed cell lines to MB and MB+VL. The results show that MB alone and MC+VL are toxic to cells, and that cells deficient in NER are not more sensitive to MC or MB+VL compared to NER proficient normal cells. In fact, the NER deficient cell lines were more resistant to MB alone compared to NER proficient normal cells. In particular, although the SV40- transformed XP-C cell line showed a significant reduction in HCR of the MB-Vl-treated reporter gene, suggesting a deficiency in the repair of MB+VL-induced DNA damage, the SV40-transformed XP-C cells were not more sensitive to MB or MB+VL. This suggests that the toxicity of human cells to MB and MB+VL results primarily from damage to cellular components other than DNA such as membrane structures including the mitochondria and lysozomes as has been reported for other photosensitizers. / Thesis / Master of Science (MSc)

The studies described in this thesis investigated the relationship between DNA repair mechanisms, aflatoxin B1 (AFB1)-induced DNA damage and carcinogenesis. Mice deficient in 8-oxoguanine glycosylase (OGG1, the rate-limiting enzyme in repair of oxidized guanine), mice heterozygous for OGG1, and wild type mice, were exposed to a single tumourigenic dose (50 mg/kg) of AFB1. Neither ogg1 genotype nor AFB1 treatment affected levels of oxidized guanine in lung or liver 2 h post-treatment. ogg1 (-/-) mice had increased susceptibility to AFB1 toxicity, as reflected by increased mortality within one week of AFB1 exposure. AFB1 treatment did not significantly increase lung or liver tumourigenesis compared to DMSO controls. No difference was observed between ogg1 genotypes, although a non-significant trend towards AFB1-treated ogg1 (-/-) mice being more susceptible to tumourigenicity was apparent. Overall, deletion of ogg1 did not significantly affect AFB1-induced DNA damage or tumourigenicity, suggesting that oxidized guanine may not be a major contributor to AFB1-induced tumourigenesis. The effects of AFB1 on DNA repair were assessed in p53 (a protein implicated in regulation of DNA repair) wild type and heterozygous mice. p53 (+/+) mice treated with 0, 0.2 or 1.0 ppm AFB1 for 26 weeks had increased nucleotide excision repair (NER) activities in lung and liver compared to control, which may represent an adaptive response to AFB1-derived DNA adducts. In p53 (+/-) mice, the AFB1-induced increase in NER was significantly attenuated, suggesting that loss of one allele of p53 limits the ability of NER to up-regulate in response to AFB1-induced DNA damage. Twenty-six week exposure to AFB1 did not affect base excision repair (BER) in p53 (+/+) mouse lung or liver compared to control. BER was significantly decreased in livers from mice exposed to 1.0 ppm AFB1 compared to those exposed to 0.2 ppm AFB1, a result that was not due to liver cell death or to altered levels of OGG1 protein. In lungs and livers of p53 (+/-) mice, BER activity was unchanged by AFB1. As such, the difference in BER response between 0.2 ppm and 1.0 ppm AFB1 treatment seen in the p53 (+/+) mice appears to be p53 dependent. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-10-17 22:24:31.577

Single cell gel electrophoresis (SCGE), more commonly known as the Comet assay, is an uncomplicated, affordable and versatile method for investigating DNA damage and repair. Existing comet–assay based methods were modified and applied in this study in order to examine the effects of different perturbations on the DNA repair capacity of different samples. Mitochondrial functioning has a vast effect on overall cell physiology and does not simply involve the production of energy in the form of ATP that sustains common biological processes, but is also associated with important cellular occurrences such as apoptosis and ROS production. It is suggested that a change in mitochondrial function may lead to extensive ROS production which may negatively affect macromolecules, including proteins involved in DNA repair pathways, and impaired energy formation which in turn may hamper the proper occurrence of energy driven processes. Complex I and ?III knock–down systems established in 143B cells are used to investigate the effect that perturbations of the energy metabolism may have on DNA repair capacity. Metallothioneins (MTs) are known to play an imperative role in trace element homeostasis and detoxification of metals and are effective ROS scavengers. The prooxidant environment that heavy metal imbalance causes may result in mutagenesis and transformation through DNA damage. It is suggested that an imbalance in the metal homeostasis caused by MT knock–out may create an environment favourable for DNA damage formation and at the same time impair DNA repair pathways. Because of the multi–functionality and involvement of metallothioneins in such a wide variety of biological processes, it was considered interesting and essential to extend the investigation on the effect of the absence of metallothioneins on DNA repair. A metallothionein I and ?II knock–out mouse model is employed to determine the effect of MT knock–out on DNA repair capacity. It was clear from the results obtained that transfection of cells, as used to investigate a perturbation in the energy metabolism in 143B cells, has an impairing effect on DRC. It was also confirmed that metallothioneins play an important and diverse role in cell biology since the absence thereof inhibits both BER and NER. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Single cell gel electrophoresis (SCGE), more commonly known as the Comet assay, is an uncomplicated, affordable and versatile method for investigating DNA damage and repair. Existing comet–assay based methods were modified and applied in this study in order to examine the effects of different perturbations on the DNA repair capacity of different samples. Mitochondrial functioning has a vast effect on overall cell physiology and does not simply involve the production of energy in the form of ATP that sustains common biological processes, but is also associated with important cellular occurrences such as apoptosis and ROS production. It is suggested that a change in mitochondrial function may lead to extensive ROS production which may negatively affect macromolecules, including proteins involved in DNA repair pathways, and impaired energy formation which in turn may hamper the proper occurrence of energy driven processes. Complex I and ?III knock–down systems established in 143B cells are used to investigate the effect that perturbations of the energy metabolism may have on DNA repair capacity. Metallothioneins (MTs) are known to play an imperative role in trace element homeostasis and detoxification of metals and are effective ROS scavengers. The prooxidant environment that heavy metal imbalance causes may result in mutagenesis and transformation through DNA damage. It is suggested that an imbalance in the metal homeostasis caused by MT knock–out may create an environment favourable for DNA damage formation and at the same time impair DNA repair pathways. Because of the multi–functionality and involvement of metallothioneins in such a wide variety of biological processes, it was considered interesting and essential to extend the investigation on the effect of the absence of metallothioneins on DNA repair. A metallothionein I and ?II knock–out mouse model is employed to determine the effect of MT knock–out on DNA repair capacity. It was clear from the results obtained that transfection of cells, as used to investigate a perturbation in the energy metabolism in 143B cells, has an impairing effect on DRC. It was also confirmed that metallothioneins play an important and diverse role in cell biology since the absence thereof inhibits both BER and NER. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.