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1.
Folia Biol (Praha) ; 66(3): 91-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33069188

RESUMO

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.


Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/fisiologia , Genes Fúngicos , Acetiltransferases/deficiência , Acetiltransferases/genética , Acetiltransferases/fisiologia , Antifúngicos/farmacologia , Sistemas CRISPR-Cas , Cálcio/fisiologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/patogenicidade , Cátions/farmacologia , Adesão Celular , Ciclo Celular , Parede Celular/efeitos dos fármacos , Quitinases/farmacologia , Dano ao DNA , Proteínas Fúngicas/genética , Deleção de Genes , Glucana Endo-1,3-beta-D-Glucosidase/farmacologia , Hifas/crescimento & desenvolvimento , Isocitrato Desidrogenase/deficiência , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Fases de Leitura Aberta , Reprodução Assexuada , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/fisiologia , Virulência/genética
2.
Mol Cell ; 79(6): 1008-1023.e4, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32871104

RESUMO

TMPRSS2-ERG gene fusion occurs in approximately 50% of cases of prostate cancer (PCa), and the fusion product is a key driver of prostate oncogenesis. However, how to leverage cellular signaling to ablate TMPRSS2-ERG oncoprotein for PCa treatment remains elusive. Here, we demonstrate that DNA damage induces proteasomal degradation of wild-type ERG and TMPRSS2-ERG oncoprotein through ERG threonine-187 and tyrosine-190 phosphorylation mediated by GSK3ß and WEE1, respectively. The dual phosphorylation triggers ERG recognition and degradation by the E3 ubiquitin ligase FBW7 in a manner independent of a canonical degron. DNA damage-induced TMPRSS2-ERG degradation was abolished by cancer-associated PTEN deletion or GSK3ß inactivation. Blockade of DNA damage-induced TMPRSS2-ERG oncoprotein degradation causes chemotherapy-resistant growth of fusion-positive PCa cells in culture and in mice. Our findings uncover a previously unrecognized TMPRSS2-ERG protein destruction mechanism and demonstrate that intact PTEN and GSK3ß signaling are essential for effective targeting of ERG protein by genotoxic therapeutics in fusion-positive PCa.


Assuntos
Proteínas de Ciclo Celular/genética , Glicogênio Sintase Quinase 3 beta/genética , Proteínas de Fusão Oncogênica/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Proteínas Tirosina Quinases/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Tratamento Farmacológico , Proteína 7 com Repetições F-Box-WD/genética , Xenoenxertos , Humanos , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
3.
Ecotoxicol Environ Saf ; 203: 110989, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888600

RESUMO

AIM: The city of L'Aquila (central Italy) was hit by a strong earthquake in 2009 that caused the collapse of several buildings, deaths and injured people. In the following years, a great number of building sites were activated, building workers resulted intensely exposed and represent a relevant target for research on environmental mutagenesis and epidemiological surveillance. Cells of buccal mucosa are considered an appropriate site for early detecting of cytogenetic damage, since it represents the first barrier in inhalation or ingestion and can metabolize carcinogenic agents into reactive chemicals. Our study is aimed 1) at comparing the early genotoxic damage as measured by the buccal mucosa micronucleus test in two subgroups of workers defined by different occupational exposure and 2) at evaluating possible confounding variables such as lifestyle factors. METHODS AND RESULTS: A cross-sectional study was conducted in L'Aquila, on 24 outdoor workers (OWs) highly exposed on the construction sites and 26 indoor workers (IWs), all subjected to the compulsory occupational surveillance system, in the period 2017-2018. Buccal cells samples were collected and, based on the Micronucleus test, the exfoliated cells were classified in respect of nuclear changes observed. Moreover, a self-report questionnaire composed of 84 items, was administered to the workers. RESULTS: Significant differences were observed between Exp+ (OWs) and Exp- (IWs) in the number of the analyzed cells (expressed as mean value out of 1000 cells): respectively 954.46 vs 990.06 normal cells, (p < 0.001); 19.79 vs 4.95 micronucleated cells, as marker of chromosomal damage (p < 0.001); 13.93 vs 8.96 binucleated cells, as marker of failed cytokinesis (p < 0.001); 2.09 vs 1.18 karyolytic cells, as marker of cell death and damaged DNA (p < 0.05). According with a multivariate regression analysis, in addition to the job exposure (OW vs IW, beta = 12.221, p < 0.001), the only variable independently associated with an increase in Micronuclei (MNs) is the smoking habit (OWs vs IWs, beta = 6.683, p < 0.001) which, even if not associated with dust exposure, worsens cell integrity. Moreover, this worsening effect is weaker in workers not exposed to the site dust (moderation effect). Within social demographic factors, the high educational level only apparently seems to affect MNs number: even if unbalanced in favor of IWs vs OWs, this variable resulted a confounder, since its effect disappears when the interaction between these two factors is considered, because it is a covariate of smoking habit as well as of the job condition. CONCLUSION: Despite some limitation, our findings clearly confirm the role of occupational exposure as a marker of cytogenetic damage associated with MNs number in construction workers. Moreover, smoking status appears as the only other investigated factor independently associated to the outcome. The statistical model, in addition, highlights possible moderation and confounding effects, such as interaction between smoking and occupational exposure and the unbalanced school education level in workers. Micronucleus test in exfoliated buccal cells would be considered a suitable method for studying the early genotoxic damage in the construction occupational setting as well as in evaluating the efficacy of preventive practices.


Assuntos
Indústria da Construção , Dano ao DNA , Poeira , Mucosa Bucal/citologia , Exposição Ocupacional/efeitos adversos , Adulto , Estudos Transversais , Humanos , Itália , Masculino , Testes para Micronúcleos , Pessoa de Meia-Idade , Adulto Jovem
4.
Nat Commun ; 11(1): 4437, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895378

RESUMO

Efficient search for DNA damage embedded in vast expanses of the DNA genome presents one of the greatest challenges to DNA repair enzymes. We report here crystal structures of human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, that interact with the DNA containing the damaged base oxoG and the normal base G while they are nested in the DNA helical stack. The structures reveal that hOGG1 engages the DNA using different protein-DNA contacts from those observed in the previously determined lesion recognition complex and other hOGG1-DNA complexes. By applying molecular dynamics simulations, we have determined the pathways taken by the lesion and normal bases when extruded from the DNA helix and their associated free energy profiles. These results reveal how the human oxoG DNA glycosylase hOGG1 locates the lesions inside the DNA helix and facilitates their extrusion for repair.


Assuntos
DNA Glicosilases/química , Reparo do DNA , Simulação de Dinâmica Molecular , Cristalografia por Raios X , DNA/química , Dano ao DNA , Conformação Proteica
5.
Nat Commun ; 11(1): 4534, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913330

RESUMO

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proliferação de Células/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Neoplasias/genética , Estruturas R-Loop/genética , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Montagem e Desmontagem da Cromatina , Dano ao DNA , Instabilidade Genômica , Humanos , Neoplasias/patologia , Transcrição Genética
7.
Anticancer Res ; 40(9): 5159-5170, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878804

RESUMO

BACKGROUND/AIM: The aim of this study was to elucidate the possibility of sensitizing colon cancer cells to the chemotherapeutic drug SN38 and investigate its mechanism of action after combined treatment with electroporation (EP). MATERIALS AND METHODS: Cells were treated with SN38, EP and their combination for 24/48 h. The cell viability, actin cytoskeleton integrity, mitochondrial superoxide, hydroperoxides, total glutathione, phosphatidyl serine expression, DNA damages and expression of membrane ABC transporters were analyzed using conventional analytical tests. RESULTS: The combination of EP and SN38 affected cell viability and cytoskeleton integrity. This effect was accompanied by: (i) high production of intracellular superoxide and hydroperoxides and depletion of glutathione; (ii) increased DNA damage and apoptotic/ferroptotic cell death; (iii) changes in the expression of membrane ABC transporters - up-regulation of SLCO1B1 and retention of SN38 in the cells. CONCLUSION: The anticancer effect of the combined treatment of SN38 and EP is related to changes in the redox-homeostasis of cancer cells, leading to cell death via apoptosis and/or ferroptosis. Thus, electroporation has a potential to increase the sensitivity of cancer cells to conventional anticancer therapy with SN38.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Oxirredução , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Imunofluorescência , Glutationa/metabolismo , Humanos , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo
8.
Adv Exp Med Biol ; 1268: 227-253, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32918222

RESUMO

Exposure of skin cells to UV radiation results in DNA damage, which if inadequately repaired, may cause mutations. UV-induced DNA damage and reactive oxygen and nitrogen species also cause local and systemic suppression of the adaptive immune system. Together, these changes underpin the development of skin tumours. The hormone derived from vitamin D, calcitriol (1,25-dihydroxyvitamin D3) and other related compounds, working via the vitamin D receptor and at least in part through endoplasmic reticulum protein 57 (ERp57), reduce cyclobutane pyrimidine dimers and oxidative DNA damage in keratinocytes and other skin cell types after UV. Calcitriol and related compounds enhance DNA repair in keratinocytes, in part through decreased reactive oxygen species, increased p53 expression and/or activation, increased repair proteins and increased energy availability in the cell when calcitriol is present after UV exposure. There is mitochondrial damage in keratinocytes after UV. In the presence of calcitriol, but not vehicle, glycolysis is increased after UV, along with increased energy-conserving autophagy and changes consistent with enhanced mitophagy. Reduced DNA damage and reduced ROS/RNS should help reduce UV-induced immune suppression. Reduced UV immune suppression is observed after topical treatment with calcitriol and related compounds in hairless mice. These protective effects of calcitriol and related compounds presumably contribute to the observed reduction in skin tumour formation in mice after chronic exposure to UV followed by topical post-irradiation treatment with calcitriol and some, though not all, related compounds.


Assuntos
Calcitriol/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos , Vitamina D/farmacologia , Animais , Calcitriol/química , Calcitriol/metabolismo , Humanos , Vitamina D/química , Vitamina D/metabolismo , Vitaminas/química , Vitaminas/metabolismo , Vitaminas/farmacologia
9.
Anticancer Res ; 40(9): 4947-4960, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878783

RESUMO

BACKGROUND/AIM: This study aimed to investigate the anticancer effects and potential mechanisms of sclareol in a human small cell lung carcinoma (SCLC) cell line. MATERIALS AND METHODS: Cell viability was determined by the MTT assay. Cell cycle, apoptosis and caspase activity were evaluated by flow cytometry. Cell cycle and DNA damage related protein expression was determined by western blotting. In vivo evaluation of sclareol was carried out in xenografted tumor mice models. RESULTS: Sclareol significantly reduced cell viability, induced G1 phase arrest and subsequently triggered apoptosis in H1688 cells. In addition, this sclareol-induced growth arrest was associated with DNA damage as indicated by phosphorylation of H2AX, activation of ATR and Chk1. Moreover, in vivo evaluation of sclareol showed that it could inhibit tumor weight and volume in a H1688 xenograft model. CONCLUSION: Sclareol might be a novel and effective therapeutic agent for the treatment of SCLC patients.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Diterpenos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Diterpenos/uso terapêutico , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Pequenas Células do Pulmão/patologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 42(4): 485-490, 2020 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895100

RESUMO

Objective To explore the molecular mechanism of human papillomavirus subtype 16(HPV-16)E7 oncogene-induced DNA re-replication in response to DNA damage. Methods Flow cytometry was performed to examine the cell cycle changes in RPE1 E7 cells stably expressing HPV-16 E7 and its control cell RPE1 Vector after DNA damage.Immunoblotting assay was used to evaluate the early mitotic inhibitor 1(Emi1)expression in RPE1 E7 and RPE1 Vector cells with or without DNA damage.The changes of the proportion of polyploidy was detected by flow cytometry in DNA-damaged RPE1 E7 cells interfered by Emi1 small interfering RNA. Results Compared with the control cells,the proportion of polyploids in RPE1 E7 cells was significantly increased in response to DNA damage(t=6.397,P=0.0031).Emi1 protein expression was significantly increased in DNA damaged RPE1 E7 cells(t=8.241,P=0.0012).The polyploid ratio of RPE1 E7 cells was significantly reduced after Emi1 was interfered by two independent small interfering RNAs(t=2.916,P=0.0434;t=3.452,P=0.0260). Conclusion In response to DNA damage,Emi1 promoted DNA re-replication caused by HPV-16 E7.


Assuntos
Replicação do DNA , Dano ao DNA , Papillomavirus Humano 16 , Mitose , Proteínas Oncogênicas Virais
11.
Mar Environ Res ; 160: 105019, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32907733

RESUMO

Polycyclic musk compounds have been identified in environmental matrices (water, sediment and air) and in biological tissues in the last decade, yet only minimal attention has been paid to their chronic toxicity in the marine environment. In the present research, the clams Ruditapes philippinarum were exposed to 0.005, 0.05, 0.5, 5 and 50 µg/L of the fragrances Galaxolide® (HHCB) and Tonalide® (AHTN) for 21 days. A battery of biomarkers related with xenobiotics biotransformation (EROD and GST), oxidative stress (GPx, GR and LPO) and genotoxicity (DNA damage) were measured in digestive gland tissues. HHCB and AHTN significantly (p < 0.05) induced EROD and GST enzymatic activities at environmental concentrations. Both fragrances also induced GPx activity. All concentrations of both compounds induced an increase of LPO and DNA damage on day 21. Although these substances have been reported as not acutely toxic, this study shows that they might induce oxidative stress and genotoxicity in marine organisms.


Assuntos
Benzopiranos , Dano ao DNA , Ácidos Graxos Monoinsaturados , Estresse Oxidativo , Poluentes Químicos da Água , Animais , Organismos Aquáticos , Benzopiranos/toxicidade , Ácidos Graxos Monoinsaturados/toxicidade , Tetra-Hidronaftalenos , Poluentes Químicos da Água/toxicidade
12.
PLoS One ; 15(9): e0238238, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881880

RESUMO

The prognosis for patients with glioblastoma (GB) remains grim. Concurrent temozolomide (TMZ) radiation-the cornerstone of glioma control-extends the overall median survival of GB patients by only a few months over radiotherapy alone. While these survival gains could be partly attributed to radiosensitization, this benefit is greatly minimized in tumors expressing O6-methylguanine DNA methyltransferase (MGMT), which specifically reverses O6-methylguanine lesions. Theoretically, non-O6-methylguanine lesions (i.e., the N-methylpurine adducts), which represent up to 90% of TMZ-generated DNA adducts, could also contribute to radiosensitization. Unfortunately, at concentrations attainable in clinical practice, the alkylation capacity of TMZ cannot overwhelm the repair of N-methylpurine adducts to efficiently exploit these lesions. The current therapeutic application of TMZ therefore faces two main obstacles: (i) the stochastic presence of MGMT and (ii) a blunted radiosensitization potential at physiologic concentrations. To circumvent these limitations, we are developing a novel molecule called NEO212-a derivatization of TMZ generated by coupling TMZ to perillyl alcohol. Based on gas chromatography/mass spectrometry and high-performance liquid chromatography analyses, we determined that NEO212 had greater tumor cell uptake than TMZ. In mouse models, NEO212 was more efficient than TMZ at crossing the blood-brain barrier, preferentially accumulating in tumoral over normal brain tissue. Moreover, in vitro analyses with GB cell lines, including TMZ-resistant isogenic variants, revealed more potent cytotoxic and radiosensitizing activities for NEO212 at physiologic concentrations. Mechanistically, these advantages of NEO212 over TMZ could be attributed to its enhanced tumor uptake presumably leading to more extensive DNA alkylation at equivalent dosages which, ultimately, allows for N-methylpurine lesions to be better exploited for radiosensitization. This effect cannot be achieved with TMZ at clinically relevant concentrations and is independent of MGMT. Our findings establish NEO212 as a superior radiosensitizer and a potentially better alternative to TMZ for newly diagnosed GB patients, irrespective of their MGMT status.


Assuntos
Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Glioma/tratamento farmacológico , Radiossensibilizantes/uso terapêutico , Temozolomida/uso terapêutico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dacarbazina/análise , Dacarbazina/metabolismo , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Cromatografia Gasosa-Espectrometria de Massas , Glioma/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Radiossensibilizantes/análise , Radiossensibilizantes/metabolismo , Radiossensibilizantes/farmacologia , Temozolomida/análise , Temozolomida/metabolismo , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Nat Commun ; 11(1): 4868, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985517

RESUMO

DNA damage sensors DDB2 and XPC initiate global genome nucleotide excision repair (NER) to protect DNA from mutagenesis caused by helix-distorting lesions. XPC recognizes helical distortions by binding to unpaired ssDNA opposite DNA lesions. DDB2 binds to UV-induced lesions directly and facilitates efficient recognition by XPC. We show that not only lesion-binding but also timely DDB2 dissociation is required for DNA damage handover to XPC and swift progression of the multistep repair reaction. DNA-binding-induced DDB2 ubiquitylation and ensuing degradation regulate its homeostasis to prevent excessive lesion (re)binding. Additionally, damage handover from DDB2 to XPC coincides with the arrival of the TFIIH complex, which further promotes DDB2 dissociation and formation of a stable XPC-TFIIH damage verification complex. Our results reveal a reciprocal coordination between DNA damage recognition and verification within NER and illustrate that timely repair factor dissociation is vital for correct spatiotemporal control of a multistep repair process.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição TFIIH/metabolismo , Ubiquitina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Fator de Transcrição TFIIH/genética , Ubiquitinação
14.
Nat Commun ; 11(1): 4784, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963245

RESUMO

Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase µ (Polµ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polµ engaging a DSB substrate. Synapsis is mediated solely by Polµ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol µ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polµ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/química , DNA/química , Cristalografia por Raios X , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica
15.
Nat Commun ; 11(1): 4828, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973141

RESUMO

ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Núcleo Celular/metabolismo , Estresse Mecânico , Citoesqueleto de Actina , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Encéfalo , Cromatina , Citoplasma , Citoesqueleto/metabolismo , Dano ao DNA , Camundongos Knockout , Metástase Neoplásica , Neurogênese , Membrana Nuclear/metabolismo
16.
Acta Cir Bras ; 35(7): e202000706, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32876084

RESUMO

Purpose To synthesize and characterize poly(hydroxybutyrate) (PHB) and norbixin membranes to evaluate them for genotoxicity in rats and wound healing in mice by histological staining. Methods For the evaluation of genotoxicity, male rats ( Rattus novegicus ) were divided into three groups (n= 5): 5% PHB/Norbixin membrane introduced into the peritoneum by laparotomy; B - negative control; C - positive control (intraperitoneal dose of cyclophosphamide 50 mg/kg). For the evaluation of biocompatibilty, a cutaneous wound was induced on the back of males mice ( Mus musculus ) divided into two experimental treatment groups: control and membrane that underwent euthanasia after 7 and 14 days treatment. Statistical analysis ware made by One Way Anova post hoc Tukey Test (p<0.05). Results Regarding the incidence of polychromatic erythrocytes, there was no difference between negative control and 5% PHB/Norbixin membrane; however, when compared to the positive control represented by cyclophosphamide, there was a significant difference (p <0.001). As for DNA damage, the changes induced in the first 4h were repaired in 24h. In addition, the membrane was effective in abbreviating the inflammatory process and served as a scaffold due to the stimulus to reepithelialization mainly on the 7 days of treatment. Conclusion The non-genotoxic PHB/Norbixin 5% membrane presented promising results that suggest its effectiveness as a guide for tissue regeneration given its biocompatibility.


Assuntos
Carotenoides , Hidroxibutiratos , Animais , Carotenoides/toxicidade , Dano ao DNA , Hidroxibutiratos/toxicidade , Masculino , Camundongos , Poliésteres , Ratos , Cicatrização
17.
Ecotoxicol Environ Saf ; 205: 111340, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32966934

RESUMO

The increase in pesticide use in response to agricultural demands poses a risk to non-target organisms, including fish. Integrated analysis of biochemical, histopathological and genetic parameters in fish exposed to Malathion insecticide provide information on the toxicity mechanisms of this pesticide, which is classified as a probable carcinogen for humans. The present study assessed the biological responses of Colossoma macropomum after exposure to Malathion. We started determining the lethal concentration, which is the concentration capable of killing 50% of the subjects in an acute toxicity test (LC50-96 h), which was 15.77 ± 3.30 mgL-1. The fish were, then, exposed to Malathion during 96 h at a sublethal concentration, 7.30 mgL-1. Overall, we observed an increased activity of biotransformation and antioxidant enzymes, which reduced production of mitochondrial reactive oxygen species after 96 h exposure, as well as kept constant the mitochondrial respiration, Acetylcholinesterase activity and DNA damage. However, fish exposed to insecticide presented severe gill histopathological damage and increased expression of proto-oncogene ras. Taken together, the results suggest that, after four days of exposure to the Malathion, C. macropomum efficiently activates its defense mechanisms, suggesting that the basal response mechanisms are responsive. On the other hand, histopathologic damages evidenced the adverse effects of Malathion on fish, since it promoted gill necrosis and increased the expression of ras oncogene that is directly related to tumorigenesis events.


Assuntos
Antioxidantes/metabolismo , Caraciformes/metabolismo , Dano ao DNA , Inseticidas/toxicidade , Malation/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Caraciformes/genética , Brânquias/efeitos dos fármacos , Brânquias/patologia , Dose Letal Mediana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Testes de Toxicidade Aguda
18.
Bull Environ Contam Toxicol ; 105(4): 650-655, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32889604

RESUMO

Biochar (char-product), generated by pyrolyzing organic materials, is produced for the intended use of land application to promote carbon sequestration, soil improvement and crop-yield. Despite the benefits biochar applications offers, scientific probing on impacts that may result from amendments with biochar is still fragmented. In this study, impact of biochar on Eudrilus eugeniae DNA was investigated. Rice-husk biochar was applied to soil at rates up to 80% d/w and earthworms were exposed for 35-day. Impact on DNA was measured using electrophoresis-gel-extraction-method. Data obtained showed that biochar application over 25% resulted in decreased survival. Electrophoresis-gel-analysis showed that DNA decreased from 450 to 300 bp in biochar soils (p = 0.002). Biochar rates (5%-25%) induced DNA damage. The DNA showed smeared bands or tail; indicating DNA degradation and/or damage. DNA damage is a clear evidence of negative impact of biochar(s) to soil-biota; suggesting that loading of soil with biochar could have serious consequences on soil-fauna.


Assuntos
Carvão Vegetal/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Animais , Biota , Carvão Vegetal/química , Dano ao DNA , Oligoquetos/metabolismo , Oryza/metabolismo , Solo , Poluentes do Solo/análise
19.
Anticancer Res ; 40(10): 5631-5639, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988887

RESUMO

BACKGROUND/AIM: DNA damage response (DDR), wherein p21 is a cell fate determinant, is a potential cancer therapeutic target. Molecular expression during DDR was explored in ovarian clear-cell carcinoma (CCC). MATERIALS AND METHODS: CHK1, CHK2, TP53 and p21 expression in DDR was examined using immunostaining in surgical sections of CCC (n=22). Molecular alterations in two types of CCC cell lines, JHOC-5 and JHOC-9, were investigated using western blot analysis. RESULTS: Expression of DDR-associated molecules was noted in most patients. While high p21 expression was found in half of the patients, the remaining patients exhibited low p21 expression. Treatment with UC2288, a p21 inhibitor, attenuated proliferation of both cell lines, more prominently in JHOC-9, resulting in reduced viability and subsequent apoptosis. CONCLUSION: p21 Inhibitor induced cell death in cells with high p21 expression, suggesting that p21 suppression can be a therapeutic strategy to treat patients with CCC.


Assuntos
Quinase 1 do Ponto de Checagem/genética , Quinase do Ponto de Checagem 2/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Dano ao DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia
20.
Anticancer Res ; 40(10): 5399-5404, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988859

RESUMO

BACKGROUND/AIM: The aim of the present study was to investigate whether idarubicin (IDR) induces oxidative DNA damage in the presence of copper (II). MATERIALS AND METHODS: DNA damage was evaluated by pBR322 plasmid DNA cleavage. The formation of oxidative stress markers [O2 •- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] was analysed. RESULTS: IDR induced DNA damage and O2 •- and 8-OHdG generation in the presence of copper (II). CONCLUSION: IDR induced oxidative DNA damage in the presence of copper (II). Since it has been reported that the concentration of copper in the serum of cancer patients is higher than that in healthy groups, IDR-induced oxidative DNA damage in the presence of copper (II) may play an important role in anticancer therapeutic strategies.


Assuntos
Antraciclinas/farmacologia , Idarubicina/farmacologia , Neoplasias/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Antraciclinas/química , Cobre/química , Dano ao DNA/efeitos dos fármacos , Humanos , Idarubicina/química , Neoplasias/genética , Neoplasias/patologia , Espécies Reativas de Oxigênio/química , Superóxido Dismutase/genética
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