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1.
Molecules ; 28(11)2023 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-37298802

RESUMO

An important biomarker of oxidative damage in cellular DNA is the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG). Although several methods are available for the biochemical analysis of this molecule, its determination at the single cell level may provide significant advantages when investigating the influence of cell heterogeneity and cell type in the DNA damage response. to. For this purpose, antibodies recognizing 8-oxodG are available; however, detection with the glycoprotein avidin has also been proposed because of a structural similarity between its natural ligand biotin and 8-oxodG. Whether the two procedures are equivalent in terms of reliability and sensitivity is not clear. In this study, we compared the immunofluorescence determination of 8-oxodG in cellular DNA using the monoclonal antibody N45.1 and labeling using avidin conjugated with the fluorochrome Alexa Fluor488 (AF488). Oxidative DNA damage was induced in different cell types by treatment with potassium bromate (KBrO3), a chemical inducer of reactive oxygen species (ROS). By using increasing concentrations of KBrO3, as well as different reaction conditions, our results indicate that the monoclonal antibody N45.1 provides a specificity of 8-oxodG labeling greater than that attained with avidin-AF488. These findings suggest that immunofluorescence techniques are best suited to the in situ analysis of 8-oxodG as a biomarker of oxidative DNA damage.


Assuntos
Avidina , Desoxiguanosina , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Desoxiguanosina/metabolismo , Reprodutibilidade dos Testes , Dano ao DNA , Estresse Oxidativo , Biomarcadores/metabolismo , Anticorpos Monoclonais/metabolismo , DNA/química
2.
Int J Mol Sci ; 23(13)2022 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-35806061

RESUMO

The p21CDKN1A protein is an important player in the maintenance of genome stability through its function as a cyclin-dependent kinase inhibitor, leading to cell-cycle arrest after genotoxic damage. In the DNA damage response, p21 interacts with specific proteins to integrate cell-cycle arrest with processes such as transcription, apoptosis, DNA repair, and cell motility. By associating with Proliferating Cell Nuclear Antigen (PCNA), the master of DNA replication, p21 is able to inhibit DNA synthesis. However, to avoid conflicts with this process, p21 protein levels are finely regulated by pathways of proteasomal degradation during the S phase, and in all the phases of the cell cycle, after DNA damage. Several lines of evidence have indicated that p21 is required for the efficient repair of different types of genotoxic lesions and, more recently, that p21 regulates DNA replication fork speed. Therefore, whether p21 is an inhibitor, or rather a regulator, of DNA replication and repair needs to be re-evaluated in light of these findings. In this review, we will discuss the lines of evidence describing how p21 is involved in DNA repair and will focus on the influence of protein interactions and p21 stability on the efficiency of DNA repair mechanisms.


Assuntos
Dano ao DNA , Reparo do DNA , Ciclo Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fase S
3.
Toxics ; 10(3)2022 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-35324756

RESUMO

Pyrethroids are neurotoxicants for animals, showing a pattern of toxic action on the nervous system. Flumethrin, a synthetic pyrethroid, is used against ectoparasites in domestic animals, plants, and for public health. This compound has been shown to be highly toxic to bees, while its effects on other animals have been less investigated. However, in vitro studies to evaluate cytotoxicity are scarce, and the mechanisms associated with this effect at the molecular level are still unknown. This study aimed to investigate the oxidative stress and cell death induction in SH-SY5Y neuroblastoma cells in response to flumethrin exposure (1-1000 µM). Flumethrin induced a significant cytotoxic effect, as evaluated by MTT and LDH leakage assays, and produced an increase in the biomarkers of oxidative stress as reactive oxygen species and nitric oxide (ROS and NO) generation, malondialdehyde (MDA) concentration, and caspase-3 activity. In addition, flumethrin significantly increased apoptosis-related gene expressions (Bax, Casp-3, BNIP3, APAF1, and AKT1) and oxidative stress and antioxidative (NFκB and SOD2) mediators. The results demonstrated, by biochemical and gene expression assays, that flumethrin induces oxidative stress and apoptosis, which could cause DNA damage. Detailed knowledge obtained about these molecular changes could provide the basis for elucidating the molecular mechanisms of flumethrin-induced neurotoxicity.

4.
Int J Mol Sci ; 22(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205418

RESUMO

Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-ß-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.


Assuntos
Cromatina/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Linhagem Celular , Histonas/metabolismo , Humanos , Estruturas R-Loop
5.
Carcinogenesis ; 41(3): 257-266, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31504229

RESUMO

Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant disorder characterized by intellectual disability, skeletal abnormalities, growth deficiency and an increased risk of tumors. RSTS is predominantly caused by mutations in CREBBP or EP300 genes encoding for CBP and p300 proteins, two lysine acetyl-transferases (KAT) playing a key role in transcription, cell proliferation and DNA repair. However, the efficiency of these processes in RSTS cells is still largely unknown. Here, we have investigated whether pathways involved in the maintenance of genome stability are affected in lymphoblastoid cell lines (LCLs) obtained from RSTS patients with mutations in CREBBP or in EP300 genes. We report that RSTS LCLs with mutations affecting CBP or p300 protein levels or KAT activity, are more sensitive to oxidative DNA damage and exhibit defective base excision repair (BER). We have found reduced OGG1 DNA glycosylase activity in RSTS compared to control cell extracts, and concomitant lower OGG1 acetylation levels, thereby impairing the initiation of the BER process. In addition, we report reduced acetylation of other BER factors, such as DNA polymerase ß and Proliferating Cell Nuclear Antigen (PCNA), together with acetylation of histone H3. We also show that complementation of CBP or p300 partially reversed RSTS cell sensitivity to DNA damage. These results disclose a mechanism of defective DNA repair as a source of genome instability in RSTS cells.


Assuntos
Proteína de Ligação a CREB/genética , DNA Glicosilases/genética , Proteína p300 Associada a E1A/genética , Síndrome de Rubinstein-Taybi/genética , Acetilação , Carcinogênese/genética , Linhagem Celular Tumoral , Dano ao DNA/genética , Reparo do DNA/genética , Humanos , Mutação , Estresse Oxidativo/genética , Fenótipo , Síndrome de Rubinstein-Taybi/patologia
6.
Int J Mol Sci ; 20(22)2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31731696

RESUMO

The importance of determining at the cellular level the formation of DNA-protein complexes after radiation-induced lesions to DNA is outlined by the evidence that such interactions represent one of the first steps of the cellular response to DNA damage. These complexes are formed through recruitment at the sites of the lesion, of proteins deputed to signal the presence of DNA damage, and of DNA repair factors necessary to remove it. Investigating the formation of such complexes has provided, and will probably continue to, relevant information about molecular mechanisms and spatiotemporal dynamics of the processes that constitute the first barrier of cell defense against genome instability and related diseases. In this review, we will summarize and discuss the use of in situ procedures to detect the formation of DNA-protein complexes after radiation-induced DNA damage. This type of analysis provides important information on the spatial localization and temporal resolution of the formation of such complexes, at the single-cell level, allowing the study of heterogeneous cell populations.


Assuntos
Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Animais , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/química , Imunofluorescência , Humanos
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