DNA Base Excision Repair System Research Articles

Role of metformin on base excision repair pathway in p53 wild-type H2009 and HepG2 cancer cells.

The antidiabetic agent metformin was shown to further possess chemopreventive and chemotherapeutic effects against cancer. Despite the advances, the underlying molecular mechanisms involved in decreasing tumor formation are still unclear. The understanding of the participation of oxidative stress in the action mechanism of metformin and its related effects on p53 and on DNA base excision repair (BER) system can help us to get closer to solve metformin puzzle in cancer. We investigated the effects of metformin in HepG2 and H2009 cells, verifying cytotoxicity, oxidative stress, antioxidant status, and DNA BER system. Our results showed metformin induced oxidative stress and reduced antioxidant capacity. Also, metformin treatment with hydrogen peroxide (H2O2) enhanced these effects. Although DNA BER enzyme activities were not changed accordantly together by metformin as a single agent or in combination with H2O2, activated p53 was decreased with increased oxidative stress in H2009 cells. Our study on the relationship between metformin/reactive oxygen species and DNA BER system in cancer cells would be helpful to understand the anticancer effects of metformin through cellular signal transduction pathways. These findings can be a model of the changes on oxidative stress that reflects p53's regulatory role on DNA repair systems in cancer for the future studies.

AP Endonuclease 1 as a Key Enzyme in Repair of Apurinic/Apyrimidinic Sites.

Human apurinic/apyrimidinic endonuclease 1 (APE1) is one of the key participants in the DNA base excision repair system. APE1 hydrolyzes DNA adjacent to the 5'-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3'-hydroxyl group and a 5'-deoxyribose phosphate moiety. APE1 exhibits 3'-phosphodiesterase, 3'-5'-exonuclease, and 3'-phosphatase activities. APE1 was also identified as a redox factor (Ref-1). In this review, data on the role of APE1 in the DNA repair process and in other metabolic processes occurring in cells are analyzed as well as the interaction of this enzyme with DNA and other proteins participating in the repair system.

Investigation of micronucleus induction in MTH1 knockdown cells exposed to UVA, UVB or UVC

The longer wave parts of UVR can increase the production of reactive oxygen species (ROS) which can oxidize nucleotides in the DNA or in the nucleotide pool leading to mutations. Oxidized bases in the DNA are repaired mainly by the DNA base excision repair system and incorporation of oxidized nucleotides into newly synthesized DNA can be prevented by the enzyme MTH1. Here we hypothesize that the formation of several oxidized base damages (from pool and DNA) in close proximity, would cause a high number of base excision repair events, leading to DNA double strand breaks (DSB) and therefore giving rise to cytogenetic damage. If this hypothesis is true, cells with low levels of MTH1 will show higher cytogenetic damage after the longer wave parts of UVR. We analyzed micronuclei induction (MN) as an endpoint for cytogenetic damage in the human lymphoblastoid cell line, TK6, with a normal and a reduced level of MTH1 exposed to UVR. The results indicate a higher level of micronuclei at all incubation times after exposure to the longer wave parts of UVR. There is no significant difference between wildtype and MTH1-knockdown TK6 cells, indicating that MTH1 has no protective role in UVR-induced cytogenetic damage.This indicates that DSBs induced by UV arise from damage forms by direct interaction of UV or ROS with the DNA rather than through oxidation of dNTP.

Sensitizing effect of silencing Ape1/Ref-1 on doxorubicin-induced apoptosis in human carcinoma cells

Ape1/Ref-1 is a multifunctional protein with major functions in DNA base excision repair system and redox regulation of several transcription factors. However, overexpression of Ape1/Ref-1 is observed in many types of malignant cancers, leading to point that this protein might be a molecular candidate for anticancer drug treatment. A doxorubicin (DOX) has been widely applied in the chemotherapy of solid tumors including lung. Cytotoxification of DOX is targeting on damage to cellular components, particularly DNA molecules, via generation of reactive oxygen species (ROS). Using specific suppression by short hairpin RNA (shRNA)-based viral vector, an important role of Ape1/Ref-1 on DOX resistance in human lung cancer H1299 cell was investigated in this study. Our data revealed that the stable Ape1/Ref-1 shRNA knockdown had higher susceptibility to DOX compared to the wild type. In accordance, intracellular ROS level was notably accelerated in the Ape1/Ref-1 shRNA defect rather than the wild type. Strikingly, our observation represented that in presence of DOX Ape1/Ref-1 deficiency probably stimulated double strand breaks to DNA, detected by neutral comet assay and gamma-H2AX immunoassay, sensitive methods of genotoxicity. Furthermore, our result showed that DOX-induced apoptosis was also correlated with increase in oxygen radicals-generated damage to DNA. We can conclude that Ape1/Ref1-targeted silencing via shRNA-based interference might be a promising molecular strategy to improve effectiveness of DOX-induced apoptosis in treatment of human lung cancer cells, through induction of oxidative insult. This emphasizes that Ape1/Ref-1 might be a prominent therapeutic target in cancer therapy.

Poly(ADP-ribose) polymerase-1 is involved in the neuronal death induced by quinolinic acid in rats

Reactive oxygen and nitrogen species formation leads to DNA damage in animals treated with quinolinic acid. Poly(ADP-ribose) polymerase-1 (PARP-1) is a protein involved in the DNA base excision repair system. Its overactivation promotes cellular energy deficit and necrosis. Here, we evaluated the effect of PJ-34, a potent inhibitor of PARP-1, on the neuronal damage induced by quinolinic acid. Animals were administered with PJ-34 (10 mg/kg, i.p.), 1 h before and 1 h after a striatal infusion of 1 microl of quinolinic acid (240 nmol). PJ-34 clearly attenuated the circling behavior produced by quinolinic acid and completely prevented the histological damage induced by the toxin. The protective effect of PJ-34 suggests that PARP-1 activation is playing an active role in the neuronal death induced by quinolinic acid.

Association of the hOGG1 Ser326Cys polymorphism with sporadic amyotrophic lateral sclerosis

Amyotropic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease causing the loss of motoneurons of the brain and the spinal cord. The etiology of ALS is still uncertain, but males are at increased risk for the disease than females. Several studies have suggested that motoneurons in ALS might be subjected to the double insult of increased DNA oxidative damage and deficiencies in DNA repair systems. Particularly, increased levels of 8-oxoguanine and impairments of the DNA base excision repair system have been observed in neurons of ALS patients. There is evidence that the Ser326Cys polymorphism of the human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene is associated with a reduced DNA repair activity. To evaluate the role of the hOGG1 Ser326Cys polymorphism in sporadic ALS (sALS), we screened 136 patients and 129 matched controls. In the total population, we observed association between both the Cys326 allele (p=0.02) and the combined Ser326Cys+Cys326Cys genotype (OR=1.65, 95% CI=1.06-2.88) and increased risk of disease. After stratification by gender, the Cys326 allele (p=0.01), both the Ser326Cys genotype (OR=2.14, 95% CI=1.09-4.19) and the combined Ser326Cys+Cys326Cys genotype (OR=2.15, 95% CI=1.16-4.01) were associated with sALS risk only in males. No significant association between the Ser326Cys polymorphism and disease phenotype, including age and site of onset and disease progression, was observed. Present results suggest a possible involvement of the hOGG1 Ser326Cys polymorphism in sALS pathogenesis.

Gene-targeted mice lacking the Trex1 (DNase III) 3'-->5' DNA exonuclease develop inflammatory myocarditis.

TREX1, originally designated DNase III, was isolated as a major nuclear DNA-specific 3'-->5' exonuclease that is widely distributed in both proliferating and nonproliferating mammalian tissues. The cognate cDNA shows homology to the editing subunit of the Escherichia coli replicative DNA polymerase III holoenzyme and encodes an exonuclease which was able to serve a DNA-editing function in vitro, promoting rejoining of a 3' mismatched residue in a reconstituted DNA base excision repair system. Here we report the generation of gene-targeted Trex1(-/-) mice. The null mice are viable and do not show the increase in spontaneous mutation frequency or cancer incidence that would be predicted if Trex1 served an obligatory role of editing mismatched 3' termini generated during DNA repair or DNA replication in vivo. Unexpectedly, Trex1(-/-) mice exhibit a dramatically reduced survival and develop inflammatory myocarditis leading to progressive, often dilated, cardiomyopathy and circulatory failure.

TSH controls Ref-1 nuclear translocation in thyroid cells.

Ref-1 (called also APE) is a bifunctional protein playing a role in a large variety of cell functions. It is a major member of the DNA base excision repair system. Moreover, through reduction of cysteine residues, Ref-1 controls the activity of several transcription factors. It has been previously demonstrated that TSH up-regulates Ref-1 gene expression in thyroid cells. By using the rat FRTL-5 cell line, we demonstrate that TSH controls Ref-1 intracellular localization. Western blot experiments indicate that addition of TSH to the culture medium increases the Ref-1 cytoplasm-to-nucleus translocation. This phenomenon occurs at early times of TSH stimulation and is not dependent on protein neosynthesis. The Ref-1 cellular compartmentalization was also investigated in human thyroid tumors. A Ref-1 nuclear/cytoplasmic ratio difference between normal and cancerous thyroid tissues was observed. These results suggest that Ref-1 localization may have a critical role in the control of thyroid cell functions.