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Introduction: In Japan, telemedicine has gradually expanded due to deregulation in response to the COVID-19 pandemic. However, its current status remains unclear, as it is primarily provided by general practitioners. Meanwhile, telemedicine has begun to be utilized for low-dose estrogen-progestin (LEP) prescriptions for dysmenorrhea. Methods: We conducted a retrospective analysis of medical record data from two gynecology clinics and performed an exploratory evaluation between a group that combined telemedicine and in-person visits during the initial 6 months of LEP treatment, and another group with only in-person visits. Results: After propensity score matching, 89 and 83 patients were eligible for the telemedicine and in-person groups, respectively, with 53 patients in both. The characteristics of both groups were similar after matching. There were no significant differences in the probability of abnormal uterine bleeding during the first 6 months of treatment (25% and 43% in each group; p = 0.064), side effects, or treatment efficacy between the two groups. The withdrawal rate at 6 months was significantly higher in the telemedicine group than in the in-person group (13% and 0%, p = 0.013). The average copayment for patients who covered 30% of the total cost was also significantly higher in the telemedicine group after 1 and 3 months of LEP prescription. Conclusion: The appropriate combination of telemedicine and in-person visits is currently employed in hospital visits, which does not differ significantly from in-person visits. Given the retrospective nature of this study and the limited number of cases, further investigation is necessary in the future.
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Background: Some papers have highlighted a possible causal relationship between the onset of ischemic stroke and weather conditions. This study aimed to elucidate the onset mechanism of cerebral infarction from a meteorological approach. We focused on the atmospheric pressure distribution patterns (APDPs). Methods: The subjects are 221 cases diagnosed as cardiogenic cerebral embolism (Group A) and 612 cases diagnosed as atherosclerotic cerebral thrombosis (Group B). We investigated the APDP on the date closest to the date and time of onset of cerebral infarction in each patient on the website and chose the most similar one from the reported 11 APDPs. Groups A and B were compared for clinical characteristics and the appearance rate of each APDP in each group. Results: The clinical characteristics of Groups A and B were consistent with some previously reported clinical characteristics of cerebral embolism and cerebral thrombosis except for smoking. The appearance rate of the other high-pressure type, which cannot be classified as either the anticyclone belt type or the migratory anticyclone type, in Group B was statistically significantly higher than that in Group A, and the appearance rate of the anticyclone belt type in Group A was statistically significantly higher than that in Group B (p < 0.05, Fisher's exact probability method, respectively). Conclusions: Cerebral embolism and cerebral thrombosis exhibited significant differences in APDPs on the day of onset. Dehydration particularly in the other high-pressure type or in the anticyclone belt type should be prevented. Further investigation should focus on the other meteorological factors.
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Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs--zebrafish ZfL2-2 and human L1--in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.
Assuntos
Reparo do DNA , Elementos Nucleotídeos Longos e Dispersos , Mutagênese Insercional , Animais , Galinhas , Enzimas Reparadoras do DNA/metabolismo , Células HeLa , Humanos , Modelos Genéticos , Peixe-ZebraRESUMO
In higher animal cells, the principal limitation of gene-targeting technology is the extremely low efficiency of targeted integration, which occurs three to four orders of magnitude less frequently than random integration. Assuming that random integration mechanistically involves non-homologous end-joining (NHEJ), inactivation of this pathway should reduce random integration and may enhance gene targeting. To test this possibility, we examined the frequencies of random and targeted integration in NHEJ-deficient chicken DT40 and human Nalm-6 cell lines. As expected, loss of NHEJ resulted in drastically reduced random integration in DT40 cells. Unexpectedly, however, this was not the case for Nalm-6 cells, indicating that NHEJ is not the sole mechanism of random integration. Nevertheless, we present evidence that NHEJ inactivation can lead to enhanced gene targeting through a reduction of random integration and/or an increase in targeted integration by homologous recombination. Most intriguingly, our results show that, in the absence of functional NHEJ, random integration of targeting vectors occurs more frequently than non-targeting vectors (harboring no or little homology to the host genome), implying that suppression of NHEJ-independent random integration events is needed to greatly enhance gene targeting in animal cells.
Assuntos
Marcação de Genes/métodos , Recombinação Genética , Animais , Linhagem Celular Tumoral , Galinhas/genética , DNA/metabolismo , DNA Ligases/genética , Deleção de Genes , Vetores Genéticos , HumanosRESUMO
KU70(-/-) and DNA-PKcs(-/-/-)chicken DT40 cells are reportedly highly sensitive to the DNA topoisomerase II inhibitor etoposide. Here we report that KU70 and DNA-PKcs unexpectedly function together during the induction of apoptosis after exposure to high levels of etoposide. In the presence of 100 microM etoposide, apoptosis was induced within 1 h in wild type DT40 cells but not in KU70(-/-) and DNA-PKcs(-/-/-) cells. In addition, the DNA-PK inhibitors NU7026 and wortmannin, as well as the caspase inhibitor Z-VAD-FMK, inhibited etoposide-induced apoptosis in wild type cells. Although Artemis(-/-) cells also showed defects in the etoposide-induced apoptosis, the other mutants defective in nonhomologous end-joining (NHEJ), LIG4(-/-), XRCC4(-), and XLF(-/-) cells were capable to induce apoptosis. When cells were treated with high doses of etoposide, the chromatin binding of DNA-PKcs was impaired by deletion of KU70 but not of Artemis, suggesting that KU70 acts upstream of DNA-PKcs and Artemis acts downstream of DNA-PKcs in the apoptotic pathway like the NHEJ pathway. These results suggest that the proteins involved in the early stage of NHEJ pathway including Artemis but not the downstream factors decide the cell fate by selecting apoptosis or DNA repair according to the degree of DNA damage.
Assuntos
Antígenos Nucleares/metabolismo , Apoptose , Quebras de DNA de Cadeia Dupla , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Cromatina/enzimologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Proteína Quinase Ativada por DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Etoposídeo/farmacologia , Autoantígeno Ku , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Recombinação Genética/efeitos dos fármacosRESUMO
Although heat shock proteins (HSP) are well known to contribute to thermotolerance, they only play a supporting role in the phenomenon. Recently, it has been reported that heat sensitivity depends on heat-induced DNA double-strand breaks (DSB), and that thermotolerance also depends on the suppression of DSB formation. However the critical elements involved in thermotolerance have not yet been fully identified. Heat produces DSB and leads to cell death through denaturation and dysfunction of heat-labile repair proteins such as DNA polymerase-beta (Pol beta). Here the authors show that thermotolerance was partially suppressed in Pol beta(-/-) mouse embryonic fibroblasts (MEF) when compared to the wild-type MEF, and was also suppressed in the presence of the HSP inhibitor, KNK437, in both cell lines. Moreover, the authors found that heat-induced gamma H2AX was suppressed in the thermotolerant cells. These results suggest that Pol beta at least contributes to thermotolerance through its reactivation and stimulation by Hsp27 and Hsp70. In addition, it appears possible that fewer DSB were formed after a challenging heat exposure because preheat-induced Hsp27 and Hsp70 can rescue or restore other, as yet unidentified, heat-labile proteins besides Pol beta. The present novel findings provide strong evidence that Pol beta functions as a critical element involved in thermotolerance and exerts an important role in heat-induced DSB.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase beta/metabolismo , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Animais , Compostos Benzidrílicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Febre/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Modelos Biológicos , Pirrolidinonas/farmacologiaRESUMO
Artemis is a recently identified factor involved in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Here, we performed targeted disruption of the Artemis gene (ARTEMIS) in the human pre-B cell line Nalm-6. Unexpectedly, we found that cells lacking Artemis exhibit increased sensitivity to low doses, but not high doses, of ionizing radiation. We also show that ARTEMIS-deficient cells are hypersensitive to the topoisomerase II inhibitor etoposide, but to a much lesser extent than cells lacking DNA ligase IV, a critical component of NHEJ. Unlike DNA ligase IV-deficient cells, ARTEMIS-deficient cells were not hypersensitive to ICRF-193, a topoisomerase II inhibitor that does not stabilize topoisomerase II-DNA cleavable complexes. Collectively, our results suggest that Artemis only partially participates in the NHEJ pathway to repair DSBs in human somatic cells.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/fisiologia , Southern Blotting , Western Blotting , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/química , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA , Dicetopiperazinas , Relação Dose-Resposta à Radiação , Endonucleases , Etoposídeo/farmacologia , Citometria de Fluxo , Humanos , Piperazinas/farmacologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tolerância a Radiação , Radiação Ionizante , Recombinação Genética/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores da Topoisomerase IIRESUMO
Homologous recombination (HR) requires nuclease activities at multiple steps, but the contribution of individual nucleases to the processing of double-strand DNA ends at different stages of HR has not been clearly defined. We used chicken DT40 cells to investigate the role of flap endonuclease 1 (Fen-1) in HR. FEN-1-deficient cells exhibited a significant decrease in the efficiency of immunoglobulin gene conversion while being proficient in recombination between sister chromatids, suggesting that Fen-1 may play a role in HR between sequences of considerable divergence. To clarify whether sequence divergence at DNA ends is truly the reason for the observed HR defect in FEN-1(-/-) cells we inserted a unique I-SceI restriction site in the genome and tested various donor and recipient HR substrates. We found that the efficiency of HR-mediated DNA repair was indeed greatly diminished when divergent sequences were present at the DNA break site. We conclude that Fen-1 eliminates heterologous sequences at DNA damage site and facilitates DNA repair by HR.
Assuntos
Dano ao DNA , Endonucleases Flap/fisiologia , Recombinação Genética , Animais , Sequência de Bases , Ciclo Celular , Galinhas , DNA/química , Reparo do DNA , DNA Complementar/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/farmacologia , Endonucleases Flap/metabolismo , Citometria de Fluxo , Raios gama , Cinética , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência do Ácido Nucleico , Troca de Cromátide Irmã , Fatores de Tempo , TransfecçãoRESUMO
Gene targeting provides a powerful means for studying gene function by a reverse genetic approach. Despite recent rapid progress in gene knockdown technologies, gene knockout studies using human somatic cells will be of greater importance for analyzing the functions of human genes in greater detail. Although the frequency of gene targeting is typically very low in human cultured cells, we have recently shown that a human precursor B cell line, Nalm-6, exceptionally allows for high-efficiency gene targeting by homologous recombination. In addition, we have developed a quick and simplified method to construct gene-targeting vectors, which is applicable to all sequenced organisms as well as embryonic stem cells. The combination of the simplified vector construction technology and the highly efficient gene-knockout system using Nalm-6 cells has enabled us to disrupt virtually any locus of the human genome within one month. Our system will greatly facilitate gene-knockout studies in human cells.
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Marcação de Genes/métodos , Células Precursoras de Linfócitos B/metabolismo , Antígenos Nucleares/genética , Sequência de Bases , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Eletroporação , Vetores Genéticos , Genoma Humano , Humanos , Autoantígeno Ku , Plasmídeos/genética , Recombinação GenéticaRESUMO
Ku, the heterodimer of Ku70 and Ku86, plays crucial roles in non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. It has recently been reported that heterozygous disruption of the human KU86 locus results in haploinsufficient phenotypes, including retarded growth, increased radiosensitivity, elevated p53 levels and shortened telomeres. In this paper, however, we show that heterozygous inactivation of either the KU70 or KU86 gene does not cause any defects in cell proliferation or DSB repair in human somatic cells. Moreover, although these heterozygous cell lines express reduced levels of both Ku70 and Ku86, they appear to maintain overall genome integrity with no elevated p53 levels or telomere shortening. These results clearly indicate that Ku haploinsufficiency is not a commonly observed phenomenon in human cells. Our data also suggest that the impact of KU70/KU86 mutations on telomere metabolism varies between cell types in humans.
Assuntos
Antígenos Nucleares/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Inativação Gênica , Instabilidade Genômica , Animais , Divisão Celular , Proliferação de Células , Dimerização , Marcação de Genes , Células HCT116/patologia , Células HeLa/patologia , Humanos , Autoantígeno Ku , Camundongos , Telômero/genéticaRESUMO
Long interspersed elements (LINEs) are autonomous transposable elements that proliferate via retrotransposition, which involves reverse transcription of LINE RNAs. It is anticipated that LINE retrotransposition requires both LINE-encoded proteins and host-encoded proteins. However, identification of the host factors, their roles, and the steps at which they act on retrotransposition are poorly understood because of the lack of an appropriate genetic system to study LINE retrotransposition in a series of mutant hosts. To construct such a genetic system, we applied the retrotransposition-indicative cassette method to DT40 cells, a chicken cell line for which a variety of isogenic mutants have been established by gene targeting. Because DT40 cells are non-adherent, we utilized a selective soft agarose medium to allow the formation of colonies of cells that had undergone LINE retrotransposition. Colony formation was completely dependent on the activities of the LINE-encoded proteins and on the presence of the essential 3' region of the LINE RNA. Moreover, the selected colonies indeed carried retrotransposed LINE copies in their chromosomes, with integration features similar to those of genomic (native) LINE copies. This method thus allows the authentic selection of LINE-retrotransposed cells and the approximate recapitulation of retrotransposition events that occur in nature. Therefore, the DT40 cell system established here provides a powerful tool for the elucidation of LINE retrotransposition pathways, the host factors involved, and their roles.
Assuntos
Elementos Nucleotídeos Longos e Dispersos , Animais , Sequência de Bases , Linhagem Celular , Galinhas , DNA Recombinante/genética , Expressão Gênica , Genes Reporter , Técnicas Genéticas , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Luciferases de Vaga-Lume/genética , Modelos Genéticos , Proteínas Recombinantes/genética , Temperatura , TransfecçãoRESUMO
The Bloom syndrome helicase BLM and the tumor-suppressor protein p53 play important roles in preserving genome integrity. Here, we knock out the genes for BLM and p53 in a human pre-B-cell line, Nalm-6. We show that p53 plays an important role in cell proliferation, but not apoptosis, when BLM is absent. Intriguingly, despite the apoptotic function of p53, BLM(/)TP53(/) cells were more sensitive than either single mutant to etoposide, an anticancer agent that poisons DNA topoisomerase II. Our results suggest a direct, BLM-independent role for p53 in etoposide-induced, topoisomerase II-mediated DNA damage in human cells.
Assuntos
Síndrome de Bloom/genética , DNA Helicases/deficiência , Etoposídeo/toxicidade , Transtornos do Crescimento/genética , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Síndrome de Bloom/enzimologia , Divisão Celular , Linhagem Celular , Reparo do DNA , Deleção de Genes , Humanos , Troca de Cromátide IrmãRESUMO
In mammalian cells, DNA polymerase beta (Polbeta) functions in base excision repair. We have previously shown that Polbeta-deficient mice exhibit extensive neuronal cell death (apoptosis) in the developing nervous system and that the mice die immediately after birth. Here, we studied potential roles in the phenotype for p53, which has been implicated in DNA damage sensing, cell cycle arrest, and apoptosis. We generated Polbeta(-/-) p53(-/-) double-mutant mice and found that p53 deficiency dramatically rescued neuronal apoptosis associated with Polbeta deficiency, indicating that p53 mediates the apoptotic process in the nervous system. Importantly, proliferation and early differentiation of neuronal progenitors in Polbeta(-/-) p53(-/-) mice appeared normal, but their brains obviously displayed cytoarchitectural abnormalities; moreover, the mice, like Polbeta(-/-) p53(+/+) mice, failed to survive after birth. Thus, we strongly suggest a crucial role for Polbeta in the differentiation of specific neuronal cell types.
Assuntos
Apoptose , Diferenciação Celular , DNA Polimerase beta/deficiência , Neurônios/citologia , Neurônios/metabolismo , Proteína Supressora de Tumor p53/deficiência , Animais , Apoptose/genética , Divisão Celular , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Genótipo , Camundongos , Camundongos Knockout , Sistema Nervoso/citologia , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Fosfosserina/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Targeted gene disruption is a powerful tool for studying gene function in cells and animals. In addition, this technology includes a potential to correct disease-causing mutations. However, constructing targeting vectors is a laborious step in the gene-targeting strategy, even apart from the low efficiency of homologous recombination in mammals. Here, we introduce a quick and simplified method to construct targeting vectors. This method is based on the commercially available MultiSite Gateway technology. The sole critical step is to design primers to PCR amplify genomic fragments for homologous DNA arms, after which neither ligation reaction nor extensive restriction mapping is necessary at all. The method therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms whose genome has been sequenced. Recently, we and others have shown that the human pre-B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line has enabled rapid disruption of virtually any locus of the human genome within one month, and homozygous knockout clones lacking a human gene of interest can be created within 2-3 months. Thus, our system greatly facilitates reverse genetic studies of mammalian--particularly human--genes.
Assuntos
Embrião de Mamíferos/citologia , Marcação de Genes , Técnicas Genéticas , Vetores Genéticos , Genoma , Células-Tronco/citologia , Sequência de Bases , Linhagem Celular , DNA Ligase Dependente de ATP , DNA Ligases/genética , Primers do DNA/química , Homozigoto , Humanos , Modelos Genéticos , Dados de Sequência MolecularRESUMO
Gene targeting provides a powerful means for analyzing gene function, as exemplified by knockout mouse studies and recent work with the highly recombinogenic chicken DT40 B-lymphocyte line. In human cultured cells, however, the low frequency of gene targeting is a serious barrier to efficiently generate knockout clones. Moreover, commonly used human cell lines are karyotypically abnormal or unstable. Here, we show using promoterless targeting constructs that Nalm-6, a human pre-B ALL cell line, is highly proficient for gene targeting by homologous recombination. Indeed, the efficiency of TP53 gene targeting in Nalm-6 appears nearly two orders of magnitude higher than that in HCT116, a colon cancer cell line popularly used for gene targeting. Expression analysis revealed a lack of MSH2 expression in this cell line. As Nalm-6 has a stable neardiploid karyotype with normal p53 status, our results underscore the usefulness of Nalm-6 for gene knockout studies in humans.
Assuntos
Linfócitos B/metabolismo , Linhagem Celular , Marcação de Genes , Proteína 2 Homóloga a MutS/metabolismo , Recombinação Genética , Linhagem Celular/metabolismo , Linhagem Celular Tumoral , Aberrações Cromossômicas , Genes p53 , Humanos , Proteína 2 Homóloga a MutS/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras GenéticasRESUMO
The in vivo contribution of reactive oxygen species produced by neutrophils against Cryptococcus infection is not widely recognized. Myeloperoxidase (MPO) is a neutrophil-specific enzyme that catalyses the production of hypohalous acids such as HOCl from H2O2. This study investigated the role of MPO in immunological defence against Cryptococcus neoformans in an MPO-deficient (MPO-/-) mouse model. The survival of MPO-/- mice infected either intranasally or intravenously with C. neoformans was lower than that of identically challenged wild-type mice. The MPO-/- mice that received intranasal injection of C. neoformans had significantly larger lung fungal burdens than wild-type mice. On day 7, MPO-/- mice had a significantly higher lung concentration of interleukin (IL)-4 and lower concentrations of IL-2, IL-12p70 and interferon (IFN)-gamma than wild-type mice, suggesting a weak Th1 response in the MPO-/- mice to C. neoformans. Pathologically, the MPO-/- mice with intranasal infection showed more severe pneumonia than wild-type mice, which was associated with an increase in the levels of IL-1alpha/beta in the lungs. In addition, in MPO-/- mice, the pulmonary infection disseminated to the brain with occasional meningitis. The keratinocyte-derived cytokine (KC) level in the brain of infected MPO-/- mice was higher than that of control mice. Both intranasal and intravenous infections resulted in a higher number of fungi in the spleen of MPO-/- mice compared to wild-type, suggesting decreased resistance to C. neoformans not only in the lungs but also in the spleen in the absence of MPO. Taken together, these data suggest a major role of MPO in the response to cryptococcal infection.
Assuntos
Criptococose/imunologia , Cryptococcus neoformans/imunologia , Peroxidase/metabolismo , Pneumonia/imunologia , Animais , Sangue/microbiologia , Encéfalo/microbiologia , Contagem de Colônia Microbiana , Criptococose/microbiologia , Criptococose/patologia , Citocinas/análise , Feminino , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Meningite Criptocócica/microbiologia , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Pneumonia/microbiologia , Pneumonia/patologia , Espécies Reativas de Oxigênio/metabolismo , Baço/microbiologia , Análise de SobrevidaRESUMO
The structure-specific FEN-1 endonuclease has been implicated in various cellular processes, including DNA replication, repair and recombination. In vertebrate cells, however, no in vivo evidence has been provided so far. Here, we knocked out the FEN-1 gene (FEN1) in the chicken DT40 cell line. Surprisingly, homozygous mutant (FEN1-/-) cells were viable, indicating that FEN-1 is not essential for cell proliferation and thus for Okazaki fragment processing during DNA replication. However, compared with wild-type cells, FEN1-/- cells exhibited a slow growth phenotype, probably due to a high rate of cell death. The mutant cells were hypersensitive to methylmethane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and H2O2, but not to UV light, X-rays and etoposide, suggesting that FEN-1 functions in base excision repair in vertebrate cells.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Exodesoxirribonucleases/genética , Peróxido de Hidrogênio/farmacologia , Metanossulfonato de Metila/farmacologia , Metilnitronitrosoguanidina/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Divisão Celular/efeitos da radiação , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Galinhas , DNA/química , DNA/genética , Relação Dose-Resposta a Droga , Etoposídeo/farmacologia , Exodesoxirribonuclease V , Exodesoxirribonucleases/metabolismo , Dados de Sequência Molecular , Mutação , Análise de Sequência de DNA , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
DNA topoisomerase II (Top2) inhibitors are useful as anticancer agents, mostly by virtue of their ability to induce DNA double-strand breaks (DSBs). These DSBs are repaired almost exclusively by Rad52-dependent homologous recombination (HR) in yeast. However, we have recently shown that in vertebrate cells such lesions are primarily repaired by nonhomologous end-joining, but not HR. This finding, taken together with previous observations that disruption of RAD52 does not severely affect HR in vertebrate cells, makes it highly unlikely that Rad52 contributes to the repair of Top2-mediated DNA damage. However, in this paper we show that chicken cells lacking Rad52 do exhibit increased sensitivity to the Top2 inhibitor VP-16. Remarkably, the level of hypersensitivity of RAD52-null cells was comparable to that of RAD54-null cells, albeit only at high doses. Our data thus provide the first demonstration of a major repair defect associated with loss of Rad52 in vertebrate cells.
Assuntos
Dano ao DNA , Reparo do DNA/fisiologia , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/fisiologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Dicetopiperazinas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Mutação , Piperazinas/farmacologia , Proteína Rad52 de Recombinação e Reparo de DNA , Recombinação Genética , Inibidores da Topoisomerase IIRESUMO
Peroxynitrite is responsible for nitration in vivo, whereas myeloperoxidase can also catalyze protein nitration in the presence of high NO2(-) levels. Recent reports of myeloperoxidase-mediated enzyme inactivation or lipid peroxidation have suggested a role of myeloperoxidase in various pathological conditions. To clarify the role of myeloperoxidase in ischemic brain injury, the authors measured nitrotyrosine formation and infarct volume in myeloperoxidase-deficient or wild-type mice subjected to 2-hour focal cerebral ischemia-reperfusion. Twenty-four hours after reperfusion, infarct volume was significantly larger in myeloperoxidase-deficient mice than in wild-type mice (81 +/- 20 mm(3) vs. 52 +/- 13 mm(3), P < 0.01), and nitrotyrosine levels in the infarct region were higher in myeloperoxidase-deficient mice than in wild-type mice (13.4 +/- 6.1 microg/mg vs. 9.8 +/- 4.4 microg/mg, P = 0.13). Fourteen hours after reperfusion, the nitrotyrosine level was significantly higher in myeloperoxidase-deficient mice than in wild-type mice (3.3 +/- 2.9 microg/mg vs. 1.4 +/- 0.4 microg/mg, P < 0.05). The authors conclude that the absence of myeloperoxidase increases ischemic neuronal damage in vivo, and that the myeloperoxidase-mediated pathway is not responsible for the nitration reaction in cerebral ischemia-reperfusion.