Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.

Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.

RecA protein stimulates homologous recombination in plants.

A number of RecA-like proteins have been found in eukaryotic organisms. We demonstrate that the prokaryotic recombination protein RecA itself is capable of interacting with genomic homologous DNA in somatic plant cells. Resistance to the DNA crosslinking agent mitomycin C requires homologous recombination as well as excision repair activity. Tobacco protoplasts expressing a nucleus-targeted RecA protein were at least three times as efficient as wild-type cells in repairing mitomycin C-induced damage. Moreover, homologous recombination at a defined locus carrying an endogenous nuclear marker gene was stimulated at least 10-fold in transgenic plant cells expressing nucleus-targeted RecA. The increase in resistance to mitomycin C and the stimulation of intrachromosomal recombination demonstrate that Escherichia coli RecA protein is functional in genomic homologous recombination in plants, especially when targeted to the plant nucleus.

Aprataxin, a novel protein that protects against genotoxic stress

Ataxia-oculomotor apraxia (AOA1) is a neurological disorder with symptoms that overlap those of ataxia-telangiectasia, a syndrome characterized by abnormal responses to double-strand DNA breaks and genome instability. The gene mutated in AOA1, APTX, is predicted to code for a protein called aprataxin that contains domains of homology with proteins involved in DNA damage signalling and repair. We demonstrate that aprataxin is a nuclear protein, present in both the nucleoplasm and the nucleolus. Mutations in the APTX gene destabilize the aprataxin protein, and fusion constructs of enhanced green fluorescent protein and aprataxin, representing deletions of putative functional domains, generate highly unstable products. Cells from AOA1 patients are characterized by enhanced sensitivity to agents that cause single-strand breaks in DNA but there is no evidence for a gross defect in single-strand break repair. Sensitivity to hydrogen peroxide and the resulting genome instability are corrected by transfection with full-length aprataxin cDNA. We also demonstrate that aprataxin interacts with the repair proteins XRCC1, PARP-1 and p53 and that it co-localizes with XRCC1 along charged particle tracks on chromatin. These results demonstrate that aprataxin influences the cellular response to genotoxic stress very likely by its capacity to interact with a number of proteins involved in DNA repair.

Live cell imaging of heavy-ion-induced radiation responses by beamline microscopy

To study the dynamics of protein recruitment to DNA lesions, ion beams can be used to generate extremely localized DNA damage within restricted regions of the nuclei. This inhomogeneous spatial distribution of lesions can be visualized indirectly and rapidly in the form of radiation-induced foci using immunocytochemical detection or GFP-tagged DNA repair proteins. To analyze faster protein translocations and a possible contribution of radiation-induced chromatin movement in DNA damage recognition in live cells, we developed a remote-controlled system to obtain high-resolution fluorescence images of living cells during ion irradiation with a frame rate of the order of seconds. Using scratch replication labeling, only minor chromatin movement at sites of ion traversal was observed within the first few minutes of impact. Furthermore, time-lapse images of the GFP-coupled DNA repair protein aprataxin revealed accumulations within seconds at sites of ion hits, indicating a very fast recruitment to damaged sites. Repositioning of the irradiated cells after fixation allowed the comparison of live cell observation with immunocytochemical staining and retrospective etching of ion tracks. These results demonstrate that heavy-ion radiation-induced changes in sub-nuclear structures can be used to determine the kinetics of early protein recruitment in living cells and that the changes are not dependent on large-scale chromatin movement at short times postirradiation. © 2005 by Radiation Research Society.

Genetic risk factors and age-related macular degeneration (AMD)

Age related macular degeneration (AMD) is the leading cause of blindness in individuals older than 65 years of age. It is a multifactorial disorder and identification of risk factors enables individuals to make lifestyle choices that may reduce the risk of disease. Collaboration between geneticists, ophthalmologists, and optometrists suggests that genetic risk factors play a more significant role in AMD than previously thought. The most important genes are associated with immune system modulation and the complement system, e.g., complement factor H (CFH), factor B (CFB), factor C3, and serpin peptidase inhibitor (SERPING1). Genes associated with membrane transport, e.g., ATP-binding cassette protein (ABCR) and voltage-dependent calcium channel gamma 3 (CACNG3), the vascular system, e.g., fibroblast growth factor 2 (FGF2), fibulin-5, lysyl oxidase-like gene (LOXL1) and selectin-P (SELP), and with lipid metabolism, e.g., apolipoprotein E (APOE) and hepatic lipase (LIPC) have also been implicated. In addition, several other genes exhibit some statistical association with AMD, e.g., age-related maculopathy susceptibility protein 2 (ARMS2) and DNA excision repair protein gene (ERCC6) but more research is needed to establish their significance. Modifiable risk factors for AMD should be discussed with patients whose lifestyle and/or family history place them in an increased risk category. Furthermore, calculation of AMD risk using current models should be recommended as a tool for patient education. It is likely that AMD management in future will be increasingly influenced by assessment of genetic risk as such screening methods become more widely available. © 2013 Spanish General Council of Optometry.

Over-Expression of Endothelin 3 Sensitizes Mice to Uv-Induced Melanoma Metastasis

Melanomagenesis is influenced by environmental and genetic factors. In normal cells, ultraviolet (UV) induced photoproducts are successfully repaired by the nucleotide excision repair (NER) pathway. Mice carrying mutations in the xeroderma pigmentosum (Xp) complementation group of genes (Xpa-Xpg) lack the NER pathway and are therefore highly sensitive to UV light; however, they do not develop melanoma after UV exposure. In humans, the Endothelin 3 signaling pathway has been linked to melanoma progression and its metastatic potential. Transgenic mice that over-express Edn3 under the control of the Keratin 5 promoter (K5-Edn3) and exhibit a hyperpigmentation phenotype, were crossed with Xp deficient mice. Because melanoma is highly metastatic and many primary malignancies spread via the lymphatic system, analyzing the lymph nodes may serve useful in assessing the possible spread of tumor cells to other tissues. This study aimed to determine whether the over-expression of Edn3 is sufficient to lead to melanoma metastasis to the lymph nodes. Mice were exposed to UV radiation and analyzed for the presence of skin lesions. Mice presenting skin lesions were sacrificed and the nearest lymph nodes were excised and examined for the presence of metastasis. Mice with melanoma skin lesions presented enlarged and hyperpigmented lymph nodes. Diagnosis of melanoma was established by immunostaining with melanocyte and melanoma cell markers, and while UV radiation caused the development of skin lesions in both K5-Edn3 transgenic and control mice, only those mice carrying the K5-Edn3 transgene were found to develop melanoma metastasis to the lymph nodes. These results indicate that over-expression of Edn3 is sufficient to lead to lymph node metastasis in mice exposed to at least one dose of UV radiation.

UV-induced melanoma mouse model dependent on endothelin 3 over-expression

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.^

Avaliação de polimorfismos funcionais nos genes de reparo XRCC1, APEX1, XPD e XPF em carcinomas de células escamosas orais

Base excision repair (BER) and nucleotide excision repair (NER) pathways play critical role in maintaining genome integrity. Polymorphisms in BER and NER genes which modulate the DNA repair capacity may affect the susceptibility and prognosis of oral cancer. This study was conducted with genomic DNA from 92 patients with oral squamous cell carcinomas (OSCC) and 130 controls. The cases were followed up to explore the associations between BER and NER genes polymorphisms and the risk and prognosis of OSCC. Four single-nucleotide polymorphisms (SNPs) in XRCC1 (rs25487), APEX1 (rs1130409), XPD (rs13181) and XPF (rs1799797) genes were tested by polymerase chain reaction – quantitative real time method. The GraphPad Prism version 6.0.1 statistical software was applied for statistical analysis of association. Odds ratio (OR), hazard ratio (HR), and their 95 % confidence intervals (CIs) were calculated by logistic regression. Kaplan-Meier curve and Cox proportional hazard model were used for prognostic analysis. The presence of polymorphic variants in XRCC1, APEX1, XPD and XPF genes were not associated with an increased risk of OSCC. Gene-environment interactions with smoking were not significant for any polymorphism. The presence of polymorphic variants of the XPD gene in association with alcohol consumption conferred an increased risk of 1.86 (95% CI: 0.86 – 4.01, p=0.03) for OSCC. Only APEX1 was associated with decreased specific survival (HR 3.94, 95% CI: 1.31 – 11.88, p=0.01). These results suggest an interaction between polymorphic variants of the XPF gene and alcohol consumption. Additionally APEX1 may represent a prognostic marker for OSCC.

Avaliação do efeito da inibição do reparo de sítios abásicos na resposta inflamatória celular

Base excision repair (BER) proteins has been associated with functions beyond DNA repair. Apurynic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein involved in a plethora of cellular activities, such as redox activation of transcription factors, RNA processing and DNA repair. Some studies have described the action of the protein 8-oxoguanine (OGG1) in correcting oxidized lesions in promoters as a step in the transcription of pro-inflammatory cytokines. Despite being especially important in redox activation of transcription factors such as nuclear factor κB (NF-κB) and AP- 1, the repair activity of APE1 has not yet been associated with the inflammatory response. In this study, experimental and bioinformatic analysis approaches have been used to investigate the relationship between inhibition of the repair of abasic sites in DNA by MX, a synthetic molecule designed to inhibt the repair activity of APE1, and the modulation of the inflammatory response. The results showed that treatment of monocytes with lipopolysaccharide (LPS) and MX reduced the expression of cytokines, chemokines and toll-like receptors, and negatively regulated biological immune processes, as macrophages activation, and NF-κB and tumor necrosis factor (TNF-α) and interferon pathways, without inducing cell death. The transcriptomic analysis suggests that LPS/MX treatment induces mitochondrial dysfunction, endoplasmic reticulum stress and activation of autophagy pathways, probably activated by impairment of cellular energy and/or the accumulation of nuclear and mitochondria DNA damage. Additionally, it is proposed that the repair activity of APE1 is required for transcription of inflammatory genes by interaction with abasic sites at specific promoters and recruitment of transcriptional complexes during inflammatory signaling. This work presents a new perspective on the interactions between the BER activity and the modulation of inflammatory response, and suggests a new activity for APE1 protein as modulator of the immune response in a redox-independent manner.

Imunoexpressão das proteínas APE-1 e XRCC-1 em carcinoma epidermoide de língua oral

DNA repair systems play a critical role in protecting the human genome from damage caused by carcinogens present in the environment. Mutations in DNA repair genes may be responsible for tumor development and resistance of malignant cells to chemotherapeutic agents. The major pathway for oxidative DNA damage repair is the base excision repair pathway. The objective of this study was to investigate the immunoexpression of APE-1 and XRCC-1, which are proteins involved in DNA base excision repair and its association with clinical and histopathological parameters in oral tongue squamous cell carcinoma (OTSCC), in order to investigate a possible prognostic value for those proteins. The expression of APE-1 and XRCC-1 was evaluated semi-quantitatively by immunohistochemistry in 50 OTSCC cases. Clinical data was collected from patients’ medical charts and histopathological grading was performed for each case. Statistical analysis (Chi-square and Fisher’s exact tests; significance of 5%) was performed to determine the association between protein expressions and clinico-pathological characteristics. APE-1 was highly expressed in nucleus and cytoplasm in 56% of cases. XRCC-1 showed overexpression only in nucleus in 60% of cases. High expression of XRCC-1 was significantly associated to clinical stages I and II (P=0.02). Both proteins were not associated to other clinical parameters or histopathological grading. Our findings demonstrate that DNA base excision repair proteins APE-1 and XRCC-1 are upregulated in OTSCC, however, they are not related to clinical and histologic parameters, except for XRCC-1 association to better clinical staging. Our results indicate that the immunohistochemical expression of these proteins has no association with prognostic parameters in this tumor.

Imunoexpressão das proteínas APE-1 e XRCC-1 em carcinoma epidermoide de língua oral

DNA repair systems play a critical role in protecting the human genome from damage caused by carcinogens present in the environment. Mutations in DNA repair genes may be responsible for tumor development and resistance of malignant cells to chemotherapeutic agents. The major pathway for oxidative DNA damage repair is the base excision repair pathway. The objective of this study was to investigate the immunoexpression of APE-1 and XRCC-1, which are proteins involved in DNA base excision repair and its association with clinical and histopathological parameters in oral tongue squamous cell carcinoma (OTSCC), in order to investigate a possible prognostic value for those proteins. The expression of APE-1 and XRCC-1 was evaluated semi-quantitatively by immunohistochemistry in 50 OTSCC cases. Clinical data was collected from patients’ medical charts and histopathological grading was performed for each case. Statistical analysis (Chi-square and Fisher’s exact tests; significance of 5%) was performed to determine the association between protein expressions and clinico-pathological characteristics. APE-1 was highly expressed in nucleus and cytoplasm in 56% of cases. XRCC-1 showed overexpression only in nucleus in 60% of cases. High expression of XRCC-1 was significantly associated to clinical stages I and II (P=0.02). Both proteins were not associated to other clinical parameters or histopathological grading. Our findings demonstrate that DNA base excision repair proteins APE-1 and XRCC-1 are upregulated in OTSCC, however, they are not related to clinical and histologic parameters, except for XRCC-1 association to better clinical staging. Our results indicate that the immunohistochemical expression of these proteins has no association with prognostic parameters in this tumor.

Imunoexpressão da proteína endonuclease apurínica/apurimidínica (APE-1) em adenomas pleomórficos e carcinomas ex-adenomas pleomórficos

Introduction: Apurinic/Apyrimidinic Endonuclease 1 (APE-1) is an essential protein for DNA base excision repair (BER) pathway and regulation of redox activities. The ability of malignant cells to recognize and repair DNA damage is an important mechanism for tumor survival, and recent studies suggest that APE-1 overexpression is related to poor prognosis in some tumors. Purpose: To analyze the immunoreactivity of APE-1 in Pleomorphic Adenomas (PA) and Carcinomas Ex Pleomorphic Adenomas (CaExPA) of salivary glands. Materials and Methods: A total of 49 tumors fixed in formalin and embedded in paraffin (33 PA and 16 CaExPA) underwent immunohistochemical study by the immunoperoxidase technique. APE-1 immunoreactivity was evaluated quantitatively by the percentage of immunopositive cells. For statistical analysis a significance level of 5% (p≤ 0.05) was adopted. Results: All cases of PA and CaExPA (n=49) were positive for APE-1, however, there was a higher expression in CaExPA, with statistically significant difference (p<0.001). There was no association between APE-1 expression and tumors of major or minor salivary gland, however, not encapsulated PA (median expression = 54.2%) showed higher expression when compared to encapsulated tumors (p=0.02). APE-1 overexpression was found mainly in cases of CaExAP with lymph node metastasis (median expression = 90.3% - p=0.002) and invasive pattern (median expression = 89.9% - p=0.003), when compared to cases without metastasis and intracapsular pattern. Conclusion: This study suggests that APE-1 is deregulated in the studied tumors. The increased expression of APE-1 is associated with the absence of complete capsule in PA and it is associated with more aggressive behavior in CaExPA.

Imunoexpressão da proteína endonuclease apurínica/apurimidínica (APE-1) em adenomas pleomórficos e carcinomas ex-adenomas pleomórficos

Introduction: Apurinic/Apyrimidinic Endonuclease 1 (APE-1) is an essential protein for DNA base excision repair (BER) pathway and regulation of redox activities. The ability of malignant cells to recognize and repair DNA damage is an important mechanism for tumor survival, and recent studies suggest that APE-1 overexpression is related to poor prognosis in some tumors. Purpose: To analyze the immunoreactivity of APE-1 in Pleomorphic Adenomas (PA) and Carcinomas Ex Pleomorphic Adenomas (CaExPA) of salivary glands. Materials and Methods: A total of 49 tumors fixed in formalin and embedded in paraffin (33 PA and 16 CaExPA) underwent immunohistochemical study by the immunoperoxidase technique. APE-1 immunoreactivity was evaluated quantitatively by the percentage of immunopositive cells. For statistical analysis a significance level of 5% (p≤ 0.05) was adopted. Results: All cases of PA and CaExPA (n=49) were positive for APE-1, however, there was a higher expression in CaExPA, with statistically significant difference (p<0.001). There was no association between APE-1 expression and tumors of major or minor salivary gland, however, not encapsulated PA (median expression = 54.2%) showed higher expression when compared to encapsulated tumors (p=0.02). APE-1 overexpression was found mainly in cases of CaExAP with lymph node metastasis (median expression = 90.3% - p=0.002) and invasive pattern (median expression = 89.9% - p=0.003), when compared to cases without metastasis and intracapsular pattern. Conclusion: This study suggests that APE-1 is deregulated in the studied tumors. The increased expression of APE-1 is associated with the absence of complete capsule in PA and it is associated with more aggressive behavior in CaExPA.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.