RESUMO
High-resolution melting (HRM) analysis has been improved and applied for the first time to quantitative analysis of enzymatic reactions. By using the relative ratios of peak intensities of substrates and products, the quantitativity of conventional HRM analysis has been improved to allow detailed kinetic analysis. As an example, the ligation of sticky ends through the action of T4 DNA ligase has been kinetically analyzed, with comprehensive data on substrate specificity and other properties having been obtained. For the first time, the kinetic parameters (kobs and apparent Km ) of sticky-end ligation were obtained for both fully matched and mismatched sticky ends. The effect of ATP concentration on sticky-end ligation was also investigated. The improved HRM method should also be applicable to versatile DNA-transforming enzymes, because the only requirement is that the products have Tm values different enough from the substrates.
Assuntos
DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Pareamento Incorreto de Bases , Sequência de Bases , DNA/metabolismo , DNA Ligases/análise , DNA Ligases/metabolismo , CinéticaRESUMO
As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays.
Assuntos
Bacteriófago T4/enzimologia , Bioensaio/métodos , DNA Ligases/metabolismo , Alcanossulfonatos/química , Compostos Azo/química , Sequência de Bases , DNA Ligases/análise , DNA de Cadeia Simples/química , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sensibilidade e Especificidade , Espectrometria de FluorescênciaRESUMO
The combination of lanthanide-tagged oligonucleotide probes with inductively coupled plasma mass spectrometry (ICP-MS) as the detection technique is a novel labeling and analysis strategy for heterogeneous nucleic acid quantification assays. We describe a hybridization assay based on biotin-streptavidin affinity using lanthanide-labeled reporter probes and biotinylated capture probes. For the basic sandwich type assay, performed in streptavidin-coated microtitration wells, the limit of detection (LOD) was 7.2 fmol of DNA target, corresponding to a final concentration of 6 pM terbium-labeled probes detectable by ICP-MS after elution from the solid support. To improve the sensitivity and sequence specificity of the approach, it was combined with established molecular biological techniques, i.e., elution with a restriction endonuclease and signal and target amplification by the ligase detection reaction (LDR) and ligase chain reaction (LCR), respectively. Initial experiments showed that the enzymes facilitated the discrimination of single-base mismatches within the recognition or ligation site. Furthermore, LCR as a target amplification step resulted in a 6000-fold increase of sensitivity, and finally an LOD of 2.6 amol was achieved with an artificial double-stranded DNA target.
Assuntos
DNA/análise , Elementos da Série dos Lantanídeos/química , Espectrometria de Massas/métodos , Hibridização de Ácido Nucleico/métodos , Sondas de Oligonucleotídeos , Espectrofotometria Atômica/métodos , Animais , Bovinos , DNA/genética , DNA Ligases/análise , DNA Ligases/genética , Soroalbumina Bovina/análise , Soroalbumina Bovina/genéticaRESUMO
The internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor found in acute myeloid leukemia patients are associated with poor prognosis. Although DNA double-strand breaks (DSBs) are mainly repaired by the DNA-PK-dependent nonhomologous end-joining (NHEJ) pathway in normal mammalian cells, an alternative and less well-defined NHEJ pathway, characterized by microhomology at the repair junctions, play a role in the generation of deletions and translocations leading to cancer progression. Here we report that in FLT3/ITD-expressing cell lines and bone marrow mononuclear cells from FLT3/ITD knock-in mice, end-joining of DSBs occurs at microhomologous sequences resulting in a high frequency of DNA deletions. Strikingly, levels of Ku proteins, key components of the main NHEJ pathway, are decreased in FLT3/ITD(+) cell lines and murine FLT3/ITD bone marrow mononuclear cells. Concomitantly, levels of DNA ligase IIIα, a component of ALT NHEJ, are increased in FLT3/ITD-expressing cells. Cells treated with a FLT3 inhibitor demonstrate decreased DNA ligase IIIα and a reduction in DNA deletions, suggesting that FLT3 signaling regulates the pathways by which DSBs are repaired. Thus, therapy to inhibit FLT3/ITD signaling and/or DNA ligase IIIα may lead to repair that reduces repair errors and genomic instability.
Assuntos
Reparo do DNA , Instabilidade Genômica , Sequências Repetidas Invertidas , Leucemia Mieloide Aguda/genética , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Células da Medula Óssea , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP , DNA Ligases/análise , Progressão da Doença , Humanos , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas de XenopusRESUMO
The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label-free and DNAzyme-based strategy to detect DNA ligase activity. This novel strategy relies on the ligation-trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin-DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40â U mL(-1) and a detection limit of 0.2â U mL(-1). Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a "split DNA machine" to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01â U mL(-1).
Assuntos
Colorimetria/métodos , DNA Ligases/metabolismo , DNA Catalítico/metabolismo , DNA/química , DNA Ligase Dependente de ATP , DNA Ligases/análise , Peroxidase do Rábano Silvestre/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido NucleicoRESUMO
Failure to repair DNA double-strand breaks (DSBs) can lead to cell death or cancer. Although nonhomologous end joining (NHEJ) has been studied extensively in mammals, little is known about it in primary tissues. Using oligomeric DNA mimicking endogenous DSBs, NHEJ in cell-free extracts of rat tissues were studied. Results show that efficiency of NHEJ is highest in lungs compared to other somatic tissues. DSBs with compatible and blunt ends joined without modifications, while noncompatible ends joined with minimal alterations in lungs and testes. Thymus exhibited elevated joining, followed by brain and spleen, which could be correlated with NHEJ gene expression. However, NHEJ efficiency was poor in terminally differentiated organs like heart, kidney and liver. Strikingly, NHEJ junctions from these tissues also showed extensive deletions and insertions. Hence, for the first time, we show that despite mode of joining being generally comparable, efficiency of NHEJ varies among primary tissues of mammals.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , DNA/genética , Pulmão/metabolismo , Animais , Antígenos Nucleares/análise , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Sequência de Bases , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP , DNA Ligases/análise , DNA Ligases/genética , DNA Ligases/metabolismo , Enzimas Reparadoras do DNA/análise , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Rim/metabolismo , Autoantígeno Ku , Fígado/metabolismo , Masculino , Miocárdio/metabolismo , Ratos , Ratos WistarRESUMO
Expression of oncogenic BCR-ABL in chronic myeloid leukemia (CML) results in increased reactive oxygen species (ROS) that in turn cause increased DNA damage, including DNA double-strand breaks (DSBs). We have previously shown increased error-prone repair of DSBs by nonhomologous end-joining (NHEJ) in CML cells. Recent reports have identified alternative NHEJ pathways that are highly error prone, prompting us to examine the role of the alternative NHEJ pathways in BCR-ABL-positive CML. Importantly, we show that key proteins in the major NHEJ pathway, Artemis and DNA ligase IV, are down-regulated, whereas DNA ligase IIIalpha, and the protein deleted in Werner syndrome, WRN, are up-regulated. DNA ligase IIIalpha and WRN form a complex that is recruited to DSBs in CML cells. Furthermore, "knockdown" of either DNA ligase IIIalpha or WRN leads to increased accumulation of unrepaired DSBs, demonstrating that they contribute to the repair of DSBs. These results indicate that altered DSB repair in CML cells is caused by the increased activity of an alternative NHEJ repair pathway, involving DNA ligase IIIalpha and WRN. We suggest that, although the repair of ROS-induced DSBs by this pathway contributes to the survival of CML cells, the resultant genomic instability drives disease progression.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Ligases/fisiologia , Reparo do DNA , Exodesoxirribonucleases/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , RecQ Helicases/fisiologia , Regulação para Cima , Sobrevivência Celular , DNA Ligase Dependente de ATP , DNA Ligases/análise , Proteínas de Ligação a DNA , Progressão da Doença , Endonucleases , Exodesoxirribonucleases/análise , Instabilidade Genômica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Nucleares , Proteínas de Ligação a Poli-ADP-Ribose , RecQ Helicases/análise , Helicase da Síndrome de Werner , Proteínas de XenopusRESUMO
OBJECTIVE: The aim of this study was to examine DNA ligase activity and expression of DNA damage response pathway (DDR) genes in patients with stable angina (SA) and non-ST elevation myocardial infarction (NSTEMI) and determine whether they correlate with plaque morphology. BACKGROUND: Patients with coronary artery disease (CAD) have evidence of deoxyribonucleic acid (DNA) damage in peripheral blood mononuclear cells (PBMCs). It is unclear whether this represents excess damage or defective DNA repair activity. METHODS: DNA ligase activity and the expression of 22 DDR genes were measured in PBMCs of patients (both SA (nâ¯=â¯47) and NSTEMI (nâ¯=â¯42)) and in age and gender-matched controls (nâ¯=â¯35). Target lesion anatomical assessment was undertaken with frequency domain optical coherent tomography. RESULTS: DNA ligase activity was different across the three groups of patients (controlâ¯=â¯119⯱â¯53, NSTEMIâ¯=â¯115.6⯱â¯85.1, SAâ¯=â¯81⯱â¯55.7â¯units/g of nuclear protein; ANOVA pâ¯=â¯0.023). Pair wise comparison demonstrated that this significance is due to differences between the control and SA patients (pâ¯=â¯0.046). Genes involved in double strand break repair and nucleotide excision repair pathways were differentially expressed in patients with SA and NSTEMI. In SA patients, fibrocalcific plaques were strongly associated with GTSE1, DDB1, MLH3 and ERCC1 expression. By contrast, in NSTEMI patients the strongest association was observed between fibrous plaques and ATM and XPA expression. CONCLUSION: PBMCs from patients with CAD exhibit differences in DNA ligase activity and expression of DDR genes. Expression levels of certain DDR genes are strongly associated with plaque morphology and may play a role in plaque development and progression. Trial Registration Number URL: www.Clinicaltrials.gov; NCT02335086.
Assuntos
Angina Estável/diagnóstico por imagem , Doença da Artéria Coronariana/diagnóstico por imagem , Vasos Coronários/diagnóstico por imagem , Dano ao DNA , Enzimas Reparadoras do DNA/análise , Reparo do DNA , Leucócitos Mononucleares/patologia , Infarto do Miocárdio sem Supradesnível do Segmento ST/diagnóstico por imagem , Placa Aterosclerótica , Tomografia de Coerência Óptica , Idoso , Angina Estável/enzimologia , Angina Estável/genética , Angina Estável/patologia , Doença da Artéria Coronariana/enzimologia , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Vasos Coronários/patologia , DNA Ligases/análise , Feminino , Humanos , Leucócitos Mononucleares/enzimologia , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio sem Supradesnível do Segmento ST/enzimologia , Infarto do Miocárdio sem Supradesnível do Segmento ST/genética , Infarto do Miocárdio sem Supradesnível do Segmento ST/patologia , Valor Preditivo dos Testes , Estudos ProspectivosRESUMO
This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in the backbone of DNA and are of significant scientific interest due to their essential nature in DNA metabolism and their importance to a range of molecular biological methodologies. The electrochemical behavior of DNA at mercury and some amalgam electrodes is strongly influenced by its backbone structure, allowing a perfect discrimination between DNA molecules containing or lacking free ends. This variation in electrochemical behavior has been utilized previously for a sensitive detection of DNA damage involving the sugar-phosphate backbone breakage. Here we show that the same principle can be utilized for monitoring of a reverse process, i.e., the repair of strand breaks by action of the DNA ligases. We demonstrate applications of the electrochemical technique for a distinction between ligatable and unligatable breaks in plasmid DNA using T4 DNA ligase, as well as for studies of the DNA backbone-joining activity in recombinant fragments of E. coli DNA ligase.
Assuntos
Quebras de DNA de Cadeia Simples , DNA Ligases/metabolismo , Técnicas Eletroquímicas/métodos , DNA Ligases/análise , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Escherichia coli/enzimologiaRESUMO
Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.
Assuntos
Processamento Alternativo , DNA Ligases/genética , Meiose/genética , Espermatócitos/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Clonagem Molecular , DNA Ligase Dependente de ATP , DNA Ligases/análise , DNA Ligases/metabolismo , Reparo do DNA , DNA Complementar , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas de Ligação a Poli-ADP-Ribose , RNA Mensageiro/análise , Recombinação Genética , Mapeamento por Restrição , Análise de Sequência de DNA , Espermatócitos/citologia , Testículo/crescimento & desenvolvimento , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteínas de XenopusRESUMO
In DNA replication, the leading strand is synthesized continuously, but lagging strand synthesis requires the complex, discontinuous synthesis of Okazaki fragments, and their subsequent joining. We have used a combination of in situ extraction and dual color photobleaching to compare the dynamic properties of three proteins essential for lagging strand synthesis: the polymerase clamp proliferating cell nuclear antigen (PCNA) and two proteins that bind to it, DNA Ligase I and Fen1. All three proteins are localized at replication foci (RF), but in contrast to PCNA, Ligase and Fen1 were readily extracted. Dual photobleaching combined with time overlays revealed a rapid exchange of Ligase and Fen1 at RF, which is consistent with de novo loading at every Okazaki fragment, while the slow recovery of PCNA mostly occurred at adjacent, newly assembled RF. These data indicate that PCNA works as a stationary loading platform that is reused for multiple Okazaki fragments, while PCNA binding proteins only transiently associate and are not stable components of the replication machinery.
Assuntos
DNA Ligases/metabolismo , Replicação do DNA , DNA/metabolismo , Endonucleases Flap/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Linhagem Celular , Estruturas do Núcleo Celular/química , DNA Ligase Dependente de ATP , DNA Ligases/análise , Endonucleases Flap/análise , Camundongos , Microscopia de Fluorescência , Antígeno Nuclear de Célula em Proliferação/análise , RepliconRESUMO
DNA single-strand breaks (SSBs) are the most frequent lesions caused by oxidative DNA damage. They disrupt DNA replication, give rise to double-strand breaks and lead to cell death and genomic instability. It has been shown that the XRCC1 protein plays a key role in SSBs repair. We have recently shown in living human cells that XRCC1 accumulates at SSBs in a fully poly(ADP-ribose) (PAR) synthesis-dependent manner and that the accumulation of XRCC1 at SSBs is essential for further repair processes. Here, we show that XRCC1 and its partner protein, DNA ligase IIIalpha, localize at the centrosomes and their vicinity in metaphase cells and disappear during anaphase. Although the function of these proteins in centrosomes during metaphase is unknown, this centrosomal localization is PAR-dependent, because neither of the proteins is observed in the centrosomes in the presence of PAR polymerase inhibitors. On treatment of metaphase cells with H2O2, XRCC1 and DNA ligase IIIalpha translocate immediately from the centrosomes to mitotic chromosomes. These results show for the first time that the repair of SSBs is present in the early mitotic chromosomes and that there is a dynamic response of XRCC1 and DNA ligase IIIalpha to SSBs, in which these proteins are recruited from the centrosomes, where metaphase-dependent activation of PAR polymerase occurs, to mitotic chromosomes, by SSBs-dependent activation of PAR polymerase.
Assuntos
Centrossomo/metabolismo , Cromossomos Humanos/metabolismo , Dano ao DNA , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Linhagem Celular , Centrossomo/química , DNA Ligase Dependente de ATP , DNA Ligases/análise , Proteínas de Ligação a DNA/análise , Humanos , Peróxido de Hidrogênio/toxicidade , Mitose , Poli Adenosina Difosfato Ribose/biossíntese , Proteínas de Ligação a Poli-ADP-Ribose , Transporte Proteico , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteínas de XenopusRESUMO
Hamster EM9 cells, which lack Xrcc1 protein, have reduced levels of DNA ligase III and are defective in nuclear base excision repair. The Xrcc1 protein stabilizes DNA ligase III and may even play a direct role in catalyzing base excision repair. Since DNA ligase III is also thought to function in mitochondrial base excision repair, it seemed likely that mitochondrial DNA ligase III function would also be dependent upon Xrcc1. However, several lines of evidence indicate that this is not the case. First, western blot analysis failed to detect Xrcc1 protein in mitochondrial extracts. Second, DNA ligase III levels present in mitochondrial protein extracts from EM9 cells were indistinguishable from those seen in similar extracts from wild-type (AA8) cells. Third, the mitochondrial DNA content of both cell lines was identical. Fourth, EM9 cells displayed no defect in their ability to repair spontaneous mitochondrial DNA damage. Fifth, while EM9 cells were far more sensitive to the cytotoxic effects of ionizing radiation due to a defect in nuclear DNA repair, there was no apparent difference in the ability of EM9 and AA8 cells to restore their mitochondrial DNA to pre-irradiation levels. Thus, mitochondrial DNA ligase III function is independent of the Xrcc1 protein.
Assuntos
DNA Ligases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/enzimologia , Animais , Southern Blotting , Western Blotting , Extratos Celulares/química , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/efeitos da radiação , Cricetinae , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , DNA Ligase Dependente de ATP , DNA Ligases/análise , DNA Ligases/genética , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta à Radiação , Raios gama , Cinética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Mutação/genética , Proteínas Nucleares/análise , Proteínas de Ligação a Poli-ADP-Ribose , Tolerância a Radiação , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteínas de XenopusRESUMO
46BR is a human fibroblast strain derived from an immunodeficient young female of stunted growth. The diploid fibroblasts as well as a Simian Virus 40-transformed cell line are hypersensitive to killing by many DNA-damaging agents, exhibit a slightly increased level of spontaneous sister chromatid exchange, and show a defect in DNA ligation in vivo. 46BR is now shown to have abnormal DNA ligase I and is similar in this regard to cell lines derived from Bloom's syndrome patients. In a direct comparison, both 46BR and several Bloom's syndrome lines were found to be hypersensitive to the cytotoxic effect of simple alkylating agents, 46BR being more markedly sensitive. Bloom's syndrome lines do not exhibit the strong delay in joining of Okazaki fragments during DNA replication characteristic of 46BR. The cell line 46BR probably has a mutation in the gene encoding DNA ligase I different from those occurring in classical cases of Bloom's syndrome.
Assuntos
Síndrome de Bloom/genética , Benzamidas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , DNA Ligases/análise , Replicação do DNA , Fibroblastos/metabolismo , Humanos , Peso Molecular , Mutação , Troca de Cromátide Irmã , Ésteres do Ácido Sulfúrico/farmacologiaRESUMO
DNA ligase and DNase levels were measured in cell-free extracts from untreated mouse erythroleukemia (MEL) cells and from cells treated with dimethyl sulfoxide (Me2SO) to induce erythroid differentiation. The DNase activity present in the extracts was sensitive to inhibition by G-actin and was, therefore, presumed to be DNase I. When the MEL cells were induced to differentiate by culturing in the presence of 1.8% Me2SO for 3 or 4 days, the apparent activity of the DNA ligase decreased to approximately 12% of the value in untreated MEL cells. In contrast, the apparent DNase I activity of the extracts from Me2SO-treated cells increased over that in extracts from untreated cells by a factor of 2. The activity of acid phosphatase, a lysosomal enzyme, remained unchanged. When strain DR-10, a mutant of the MEL cells which does not undergo Me2SO-induced differentiation, was treated with Me2SO, the DNA ligase and DNase activities of extracts from these cells remained unchanged as compared to extracts from untreated DR-10 cells. Therefore, the marked increase in the level of DNA ligase activity appeared to be related to the process of differentiation in the Me2SO-treated MEL cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , DNA Ligases/análise , Desoxirribonucleases/análise , Dimetil Sulfóxido/farmacologia , Leucemia Eritroblástica Aguda/enzimologia , Polinucleotídeo Ligases/análise , Animais , Linhagem Celular , Leucemia Eritroblástica Aguda/patologia , CamundongosRESUMO
When mouse erythroleukemia (MEL) cells were induced to differentiate by growth in the presence of dimethyl sulfoxide, hexamethylene bisacetamide (HMBA), or hemin, the apparent activity of DNA ligase extractable from inducer-treated cells decreased 70 to 80% when compared to untreated cells. Earlier work had indicated that these changes did not occur in a differentiation-resistant MEL cell variant and suggested that the decrease in the level of DNA ligase activity might be related to the differentiation process. Since the MEL cells accumulate high levels of both hemoglobin-bound and non-hemoglobin-bound heme, the effect of both hemoglobin and hemin on DNA ligase activity of MEL cell extracts was tested. When cell-free extracts containing DNA ligase activity were preincubated with hemin at concentrations up to 150 microM, an 80% or greater inhibition of the DNA ligase activity resulted. The ATP-dependent DNA ligase from bacteriophage T4 was also inhibited by hemin, but the NAD-dependent DNA ligase from Escherichia coli was not sensitive to this treatment. Preincubation of these same extracts with hemoglobin at levels comparable to those present in differentiating cells did not result in inhibition of any of the ATP-dependent DNA ligases tested. Culturing the cells with dimethyl sulfoxide in the presence of imidazole resulted in a marked decrease in globin chain accumulation but did not reverse the dimethyl sulfoxide-related decrease in DNA ligase activity. These data suggest the possibility that heme or its metabolites, but not globin or hemoglobin, could serve to modify the process of DNA replication and/or repair in differentiating MEL cells via inhibition of DNA ligase activity. These data are consistent with the findings of Lo et al. (S.C. Lo, R. Aft, and G.C. Mueller, Cancer Res., 41: 864-870, 1981) which correlated the onset of differentiation-related terminal cell division in MEL cells with the levels of nonhemoglobin heme present in these cells.
Assuntos
DNA Ligases/análise , Heme/farmacologia , Leucemia Eritroblástica Aguda/enzimologia , Polinucleotídeo Ligases/análise , Acetamidas/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Diferenciação Celular , Dimetil Sulfóxido/farmacologia , Hemina/farmacologia , Imidazóis/farmacologia , Leucemia Eritroblástica Aguda/patologia , Camundongos , Peso Molecular , Células Tumorais CultivadasRESUMO
OBJECTIVES: The study investigated the expression and relationship of deoxyribonucleic acid (DNA) repair enzymes with hemodynamic and nitric oxide (NO)-mediated stress in the failing myocardium. BACKGROUND: The role of apoptosis in human heart failure is controversial. Experimental studies suggested that NO-mediated stress modulates apoptosis of the cardiac myocytes. Of note, DNA repair enzymes such as redox factor/apurinic/apyridimine endonuclease Ref-1 protein, proliferative cell nuclear antigen (PCNA), the poly (ADP-ribose) polymerase (PARP), and DNA-protein kinase (DNA-PK) determine the cell fate after the DNA damage. METHODS: Left ventricular (LV) endomyocardial biopsies from 23 patients with dilated cardiomyopathy were analyzed by immunohistochemistry. RESULTS: Terminal deoxynucleotidyltransferase-mediated biotin-dUTP nick-end labeling (TUNEL) or cleaved caspase-3 and cleaved PARP could not be detected. The number of Ref-1-positive myocytes tended to be higher in patients with LV ejection fraction (EF) < or =35% versus LV EF >35% (21.23 +/- 4.8% vs. 13.8 +/- 5.8%, p = 0.1). The PCNA (7.1 +/- 2.8% vs. 0.9 +/- 0.6%, p = 0.05) and DNA-PK expressions (39.5 +/- 5.4% vs. 8.6 +/- 5.5%, p < 0.01) were higher in patients with LVEF < or =35% vs. LVEF >35%. The PCNA, Ref-1, and DNA-PK expression correlated with the LV end-systolic wall stress (r = 0.61, p < 0.01; r = 0.52, p < 0.01; and r = 0.73, p < 0.001, respectively). In addition, the PCNA and DNA-PK expression correlated with inducible NO synthase (r = 0.41, p = 0.05, and r = 0.53, p < 0.01, respectively). CONCLUSION: In this study, apoptosis could not be detected in the failing myocardium owing to idiopathic dilated cardiomyopathy. In contrast, failing myocardium was characterized by active DNA repair that was associated with elevated LV wall stress and activation of the inducible NO synthase.
Assuntos
Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , DNA Ligases/análise , Coração/fisiopatologia , Hemodinâmica/fisiologia , Miocárdio/patologia , Adulto , Idoso , Cardiomiopatia Dilatada/genética , Feminino , Coração/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/farmacologia , Vasodilatadores/farmacologiaRESUMO
Methylating triazenes have shown marked antileukemic effects, possibly through generation of a variety of DNA adducts. Cells tolerant to O6-methylguanine due to a defect in the mismatch repair system (MRS), might become sensitive to other methyl adducts, by inhibiting the N-methylpurine repair, which requires base excision repair (BER) and poly(ADP-ribose) polymerase (PADPRP). Therefore, MRS-deficient Jurkat leukemic cells resistant to methylating triazenes, have been treated with temozolomide (TZM) and PADPRP inhibitors. Expression of PADPRP or molecules involved in the BER system [3-methylpurine-DNA glycosylase (MPG) and X-ray repair cross-complementing 1 (XRCC1)], have been explored. Cytotoxic effects of TZM associated with PADPRP inhibitors are evident shortly after treatment, suggesting that completion of cell division is not required for the lethal effect of the drug combination. Increase of PADPRP or MPG transcripts was found after treatment with TZM alone or combined with PADPRP inhibitor. XRCC1 transcript was positively modulated only in the case of drug combination. This could suggest that in the presence of PADPRP inhibitor, persistence of DNA damage triggers XRCC1 transcription. Our results suggest that association of TZM and PADPRP inhibitors might be of benefit for MRS-deficient malignancies unresponsive to the methylating agent.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Apoptose , Benzamidas/farmacologia , DNA Glicosilases , Reparo do DNA/efeitos dos fármacos , Dacarbazina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Leucemia/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases , DNA Ligases/análise , DNA Ligases/genética , Dacarbazina/farmacologia , Interações Medicamentosas , Resistencia a Medicamentos Antineoplásicos , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Células Jurkat , Leucemia/enzimologia , Leucemia/genética , N-Glicosil Hidrolases/fisiologia , RNA Mensageiro/análise , Temozolomida , Transcrição Gênica/efeitos dos fármacos , Triazenos/farmacologiaRESUMO
PURPOSE: Human DNA ligase I plays an essential role in DNA replication, recombination, and repair by catalyzing the formation of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl termini at single breaks in duplex DNA molecules. DNA ligase I is responsible for the majority of DNA ligase activity present in proliferating cells among four different forms of DNA ligases, designated DNA ligase I, II, III, and IV. In this study, we were interested in comparing DNA ligase I level in human tumors versus normal tissues and in studying whether the inhibition of DNA ligase I could lead to tumor cell death. EXPERIMENTAL DESIGN: DNA ligase I level was measured by Western immunoblot assay in various human malignant tumor specimens and benign tissues obtained from patients, in peripheral blood lymphocytes obtained from healthy donors, and in human tumors grown in nude mice. We also have designed antisense oligonucleotides (ODNs) targeting the mRNA of DNA ligase I and tested whether DNA ligase I antisense ODNs could control tumor cell growth. RESULTS: The amount of DNA ligase I enzyme in malignant tumors was considerably higher than that in benign normal tissues and peripheral blood lymphocytes. The level of DNA ligase I in human tumors grown in nude mice was also very high, and the expression of DNA ligase I appears to be constitutive during in vivo tumor development. We have observed that DNA ligase I antisense ODN inhibited tumor cell growth in a dose-response manner, but nonspecific control ODNs had little effect on the growth of the same cell lines. CONCLUSIONS: The presence of DNA ligase I at higher levels in human tumors than in benign normal tissues and normal peripheral lymphocytes suggests DNA ligase I plays more of a role in proliferating cells than in resting cells. These results, together with our finding that DNA ligase I antisense ODNs can suppress tumor cell proliferation, warrant the design and testing of human DNA ligase I inhibitors as new anticancer agents.
Assuntos
DNA Ligases/genética , Neoplasias/enzimologia , Animais , Sequência de Bases , Western Blotting , Neoplasias da Mama , Divisão Celular/fisiologia , DNA Ligase Dependente de ATP , DNA Ligases/análise , Replicação do DNA , Feminino , Humanos , Linfócitos/enzimologia , Camundongos , Camundongos Nus , Neoplasias/patologia , Neuroblastoma , Oligodesoxirribonucleotídeos Antissenso/síntese química , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Neoplasias Ovarianas , Neoplasias Pancreáticas , Valores de Referência , Sensibilidade e Especificidade , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The extensive characterization of the replicative human DNA ligase I (LigI) undertaken in the last decade demonstrated that the level of this protein strongly correlates with the rate of cell proliferation. This may allow to expand the repertoire of clinical biomarkers for the analysis of cell proliferation. We have produced a new monoclonal antibody (5H5) against LigI and exploited it as cell proliferation marker in Western blotting and immunofluorescence as well as in immunohistochemistry on paraffin tissue sections. The Western blot analysis showed that the LigI level detected by 5H5 antibody is high in all proliferating cells. On the contrary the protein is down regulated in resting human fibroblast and peripheral blood lymphocytes. Immunofluorescence analysis on cultured HeLa cells showed that 5H5 antibody labels all proliferating cells and displays the same staining pattern of BrdU in S-phase nuclei. Finally the analysis of serial sections of inflamed tonsils and NHL lymph nodes (either frozen or paraffin embedded) demonstrated that 5H5 marks the same population of cells as the Ki-67 antibody. Our results demonstrate that 5H5 antibody is a valuable tool for labeling proliferating cells that can be conveniently used in Western blotting, immunocytochemistry and immunohistochemistry.