RESUMEN
Letermovir is a human cytomegalovirus (HCMV) terminase inhibitor recently approved in the United States for prophylaxis of HCMV infection or disease in adult HCMV-seropositive recipients [R+] of an allogeneic hematopoietic stem cell transplant. In the registrational trial, the rate of clinically significant HCMV infection, defined as the development of HCMV DNAemia leading to preemptive antiviral therapy or the diagnosis of HCMV end-organ disease, through 24 weeks post-transplant, was significantly lower among subjects who received letermovir prophylaxis through 14 weeks post-transplant compared to those who received placebo. We performed independent analyses of the HCMV nucleotide sequencing data generated by next-generation sequencing from this phase 3 registrational trial of letermovir to identify viral genetic characteristics associated with virologic failure during and following letermovir prophylaxis. The pUL56 substitutions V236M, E237G, and C325W, identified at previously known resistance-associated positions, were detected in the virus of subjects who were treated with letermovir and failed letermovir prophylaxis. Several additional substitutions were detected in pUL56 and pUL89, and further characterization is needed to determine if any of these substitutions are clinically relevant. The analyses reported herein were conducted to confirm sponsor-reported drug-resistance pathways, to assess the frequency of resistance, and to better understand the risk of prophylaxis failures and treatment-emergent drug resistance.
Asunto(s)
Citomegalovirus/genética , Farmacorresistencia Viral/genética , Genómica , Proteínas Virales/genética , Proteínas Estructurales Virales/genética , Acetatos/farmacología , Sustitución de Aminoácidos , Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Endodesoxirribonucleasas/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Quinazolinas/farmacología , Trasplante de Células MadreRESUMEN
Cytomegalovirus (CMV) manifestations remain important complications after allogeneic hematopoietic stem cell transplantation (HSCT), even in the current era. Unfortunately, available anti-CMV agents, mainly viral polymerase inhibitors, have a substantial risk of myelosuppression and nephrotoxicity. Letermovir, a novel anti-CMV drug that targets the viral terminase complex, has recently been approved for the prevention of clinically significant CMV infection in adult CMV seropositive hematopoietic stem cell transplantation recipients. This molecule could become a paradigm-shifting drug in preventing CMV manifestations based on its novel mechanism of action, lack of cross-resistance with available drugs, proven efficacy in a large randomized clinical trial, and its beneficial toxicity and tolerability profile. Drug-drug interactions and the lack of any activity against other viruses are the main shortcomings of letermovir.
Asunto(s)
Acetatos/farmacología , Profilaxis Antibiótica , Antivirales/farmacología , Infecciones por Citomegalovirus/prevención & control , Citomegalovirus/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Quinazolinas/farmacología , Acetatos/efectos adversos , Acetatos/uso terapéutico , Adulto , Antivirales/efectos adversos , Antivirales/uso terapéutico , Citomegalovirus/patogenicidad , Evaluación de Medicamentos , Interacciones Farmacológicas , Farmacorresistencia Viral , Endodesoxirribonucleasas/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Quinazolinas/efectos adversos , Quinazolinas/uso terapéutico , Receptores de Trasplantes , Resultado del TratamientoRESUMEN
Herpesviruses are ubiquitous in animals and cause economic losses concomitant with many diseases. Most of the domestic animal herpesviruses are within the subfamily Alphaherpesvirinae, which includes human herpes simplex virus 1 (HSV-1). Suppression of HSV-1 replication has been reported with α-hydroxytropolones (αHTs), aromatic ring compounds that have broad bioactivity due to potent chelating activity. It is postulated that αHTs inhibit enzymes within the nucleotidyltransferase superfamily (NTS). These enzymes require divalent cations for nucleic acid cleavage activity. Potential targets include the nuclease component of the herpesvirus terminase (pUL15C), a highly conserved NTS-like enzyme that cleaves viral DNA into genomic lengths prior to packaging into capsids. Inhibition of pUL15C activity in biochemical assays by various αHTs previously revealed a spectrum of potencies. Interestingly, the most potent anti-pUL15C αHT inhibited HSV-1 replication to a limited extent in cell culture. The aim of this study was to evaluate three different αHT molecules with varying biochemical anti-pUL15C activity for a capacity to inhibit replication of veterinary herpesviruses (BoHV-1, EHV-1, and FHV-1) and HSV-1. Given the known discordant potencies between anti-pUL15C and HSV-1 replication inhibition, a second objective was to elucidate the mechanism of action of these compounds. The results show that αHTs broadly inhibit herpesviruses, with similar inhibitory effect against HSV-1, BoHV-1, EHV-1, and FHV-1. Based on immunoblotting, Southern blotting, and real-time qPCR, the compounds were found to specifically inhibit viral DNA replication. Thus, αHTs represent a new class of broadly active anti-herpesviral compounds with potential veterinary applications.
Asunto(s)
Antivirales/farmacología , Herpesviridae/efectos de los fármacos , Tropolona/análogos & derivados , Tropolona/farmacología , Replicación Viral/efectos de los fármacos , Animales , Chlorocebus aethiops , Replicación del ADN/efectos de los fármacos , ADN Viral/genética , Farmacorresistencia Viral , Endodesoxirribonucleasas/efectos de los fármacos , Herpesviridae/enzimología , Humanos , Nucleotidiltransferasas/efectos de los fármacos , Tropolona/química , Células Vero , Proteínas Virales/efectos de los fármacos , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Benzimidazole D-ribonucleosides are potent and selective inhibitors of CMV infection that have been shown to target the viral terminase, the enzyme complex responsible for viral DNA cleavage into single unit-length genomes and subsequent DNA packaging into procapsids. Here, we evaluated the viral inhibition by benzimidazole D-ribonucleosides against rat cytomegalovirus (RCMV). METHODS: Antiviral activity of compounds Cl4RB and BTCRB against RCMV was quantified by measurement of plaque formation. Yield assays and electron microscopy of thin sections was performed using RCMV-infected cells in the presence or absence of the compounds. The effects of Cl4RB and BTCRB on cleavage of concatemers was analyzed by pulsed-field gel electrophoresis. To characterize the behaviour of the antiviral compounds in a more physiological environment, a 3D cell culture model was employed where cells are embedded in an extracellular matrix using rat-tail collagen I. RESULTS: Both compounds had an inhibitory effect against RCMV-E. Electron microscopy revealed that only few virions were formed in RCMV-E infected cells in the presence of the compounds. Pulsed-field gel electrophoresis showed that DNA concatemers failed to be processed in the presence of the compounds. Yield Assays showed a comparable viral growth in the 3D vs. 2D cell culture as well as inhibition in the presence of Cl4RB or BTCRB for RCMV-E/GFP. CONCLUSIONS: These results demonstrate that the tetrahalogenated benzimidazole D-ribonucleosides are effective against RCMV-E by preventing cleavage of concatemeric DNA and nuclear egress of mature capsids.
Asunto(s)
Antivirales/farmacología , Bencimidazoles/farmacología , Infecciones por Herpesviridae/tratamiento farmacológico , Muromegalovirus/efectos de los fármacos , Nucleósidos/farmacología , Ribonucleósidos/farmacología , Animales , Antivirales/química , Bencimidazoles/química , Técnicas de Cultivo de Célula , Colágeno/química , Empaquetamiento del ADN/efectos de los fármacos , Endodesoxirribonucleasas/efectos de los fármacos , Halogenación , Infecciones por Herpesviridae/virología , Microscopía Electrónica , Modelos Biológicos , Muromegalovirus/ultraestructura , Nucleósidos/química , Ratas , Ribonucleósidos/química , Andamios del Tejido , Ensayo de Placa ViralRESUMEN
(-)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, was tested for in vitro cytotoxicity against human laryngeal epidermoid carcinoma of the larynx Hep2 cells. EGCG-induced apoptotic cell death accompanied by a change in the cell cycle. However, EGCG did not result in caspase activation, nor did a caspase inhibitor block cell death. Furthermore, EGCG caused no change in the intracellular levels of reactive oxygen species (ROS). The levels of p53 were increased in the EGCG-treated cells, with a corresponding decrease in Bcl-2 and Bid protein levels as well as an increase in the Bax level. In addition, EGCG induced the cytoplasmic release of cytochrome c from the mitochondria accompanied by a decreased mitochondrial membrane potential, and subsequently upregulated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) into the nucleus during the apoptotic process. Taken together, these findings indicate that the p53-mediated mitochondrial pathway and the nuclear translocation of AIF and EndoG play a crucial role in EGCG-induced apoptosis of human laryngeal epidermoid carcinoma Hep2 cells, which proceeds through a caspase-independent pathway.
Asunto(s)
Anticarcinógenos/farmacología , Factor Inductor de la Apoptosis/metabolismo , Carcinoma de Células Escamosas/metabolismo , Catequina/análogos & derivados , Endodesoxirribonucleasas/metabolismo , Neoplasias Laríngeas/metabolismo , Factor Inductor de la Apoptosis/efectos de los fármacos , Western Blotting , Catequina/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Endodesoxirribonucleasas/efectos de los fármacos , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Alpha-hydroxy acids (AHAs) have been widely used in cosmetic industry. However, knowledge on cytotoxicity of AHAs in human keratinocytes is limited. OBJECTIVE: Lactic acid (LA) is one of the most commonly used AHAs in skin care and peeling formulations. We investigated the antiproliferative effects of LA in a human keratinocyte cell line (HaCaT). METHODS: HaCaT cells were treated with LA at 7.5 approximately 17.5mM for various time periods. The molecular mechanisms of anti-proliferation through cell cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) stain, flow cytometry, Western blot and confocal microscopy. RESULTS: Viability of HaCaT cells decreased on exposure to LA. Flow cytometry showed apoptosis was closely related to the increase of reactive oxygen species (ROS) and calcium release, and to the decline of mitochondrial membrane potential (MMP). Western blotting showed an increase in the levels of P21, P27 and a decrease in the levels of Cyclin E, Cyclin A, and CDK 2, indicating cell cycle arrest at G1/S. The occurrence of apoptosis was proved by the increased expressions of Fas, Bax, caspase-3, -8, and -9, apoptosis-inducing factor (AIF), and endonuclease G (EndoG), and the declined expressions of Bcl-2 and Bcl-xL. In addition, the intracytosolic release of AIF, EndoG, and cytochrome c contributing to the occurrence of apoptosis was demonstrated by confocal microscopy. CONCLUSION: We demonstrated that LA had antiproliferative effect in HaCaT cell through the inhibition of cell cycle progression at G1/S, and the induction of programmed cell death through caspase-dependent and caspase-independent pathways.
Asunto(s)
Apoptosis , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citostáticos/farmacología , Queratinocitos/efectos de los fármacos , Ácido Láctico/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Calcio/metabolismo , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Ciclo Celular/fisiología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Inhibidores de Cisteína Proteinasa/farmacología , Citocromos c/efectos de los fármacos , Citocromos c/metabolismo , Endodesoxirribonucleasas/efectos de los fármacos , Endodesoxirribonucleasas/metabolismo , Humanos , Queratinocitos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS-MutL complex recruits the Uvr proteins to complete repair.
Asunto(s)
Azacitidina/administración & dosificación , Disparidad de Par Base/efectos de los fármacos , Reparación de la Incompatibilidad de ADN/fisiología , Reparación del ADN/fisiología , ADN Bacteriano/efectos de los fármacos , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/efectos de los fármacos , ADN Helicasas/deficiencia , ADN Helicasas/efectos de los fármacos , Enzimas Reparadoras del ADN/deficiencia , Enzimas Reparadoras del ADN/efectos de los fármacos , ADN Bacteriano/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/efectos de los fármacos , ADN-Citosina Metilasas/metabolismo , Relación Dosis-Respuesta a Droga , Endodesoxirribonucleasas/deficiencia , Endodesoxirribonucleasas/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/efectos de los fármacos , Mutación del Sistema de Lectura/efectos de los fármacos , Proteínas MutL , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/deficiencia , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/efectos de los fármacos , Técnicas del Sistema de Dos HíbridosRESUMEN
Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) is a small-molecule, selective, noncompetitive inhibitor directed against human IMPDH. In this report, we show that VX-944 inhibits in vitro growth of human multiple myeloma (MM) cell lines via induction of apoptosis. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs) do not protect against VX-944-induced MM cell growth inhibition. VX-944 induced apoptosis in MM cell lines with only modest activation of caspases 3, 8, and 9. Furthermore, the pan-caspase inhibitor z-VAD-fmk did not inhibit VX-944-induced apoptosis and cell death. During VX-944-induced apoptosis, expressions of Bax and Bak were enhanced, and both apoptosis-inducing factor (AIF) and endonuclease G (Endo G) were released from the mitochondria to cytosol, suggesting that VX-944 triggers apoptosis in MM cells primarily via a caspase-independent, Bax/AIF/Endo G pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin and melphalan even in the presence of BMSCs. Taken together, our data demonstrate a primarily non-caspase-dependent apoptotic pathway triggered by VX-944, thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional agents which trigger caspase activation.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Endodesoxirribonucleasas/metabolismo , Flavoproteínas/metabolismo , IMP Deshidrogenasa/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Mieloma Múltiple/patología , Compuestos Orgánicos/farmacología , Apoptosis/fisiología , Factor Inductor de la Apoptosis , Western Blotting , Células de la Médula Ósea , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Endodesoxirribonucleasas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavoproteínas/efectos de los fármacos , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Interleucina-6/metabolismo , Proteínas de la Membrana/efectos de los fármacosRESUMEN
Human Fen1 can be acetylated in vivo and in vitro resulting in reduced endonuclease and exonuclease activities in vitro. Acetylation occurs at four lysines located at the C terminus of Fen1, which is important for DNA binding. In this paper we show that Fen1 mutant proteins lacking the lysines at the C terminus have both reduced PCNA independent exonucleolytic and endonucleolytic activities. However, lysines at the C terminus are not required for PCNA stimulation of human Fen1. A double flap substrate was optimal for human Fen1 endonuclease and did not require the C-terminal lysines. Similarly, a one nucleotide 3'-overhang nick substrate was optimal for human Fen1 exonuclease and also did not require the C-terminal lysines. Finally, we found by an electromobility shift assay that human Fen1 had a different mode of binding with a double flap substrate containing a one nucleotide 3'-tail when compared to various other flap substrates. Taken together, our results confirm the double flap substrate as the likely in vivo intermediate for human Fen1 and that the C-terminal lysines are important for the endonuclease and exonuclease activities likely through DNA binding.
Asunto(s)
Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Endonucleasas de ADN Solapado , Lisina/metabolismo , Acetilación , Secuencia de Aminoácidos , Secuencia de Bases , ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Endodesoxirribonucleasas/efectos de los fármacos , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/efectos de los fármacos , Exodesoxirribonucleasas/genética , Humanos , Lisina/genética , Sustancias Macromoleculares , Magnesio/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nucleótidos/química , Nucleótidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/farmacología , Relación Estructura-ActividadRESUMEN
3-Aminobenzamide (3AB) is an inhibitor of poly (ADP-ribose) polymerase (PARP), an enzyme implicated in the maintenance of genomic integrity, which is activated in response to radiation-induced DNA strand breaks. cDNA macroarray membranes containing 1536 clones were used to characterize the gene expression profiles displayed by mouse BALB/3T3 fibroblasts (A31 cell line) in response to ionizing irradiation alone or in combination with 3AB. A31 cells in exponential growth were pre-treated with 3AB 4mM 1h before gamma-irradiation (4Gy), remaining in culture during 6h until harvesting time. A31 cells treated with 3AB alone presented a down-regulation in genes involved in protein processing and cell cycle control, while an up-regulation of genes involved in apoptosis and related to DNA/RNA synthesis and repair was verified. A31 cells irradiated with 4Gy displayed 41 genes differentially expressed, being detected a down-regulation of genes involved in protein processing and apoptosis, and genes controlling the cell cycle. Concomitantly, another set of genes for protein processing and related to DNA/RNA synthesis and repair were found to be up-regulated. A positive or negative interaction effect between 3AB and radiation was verified for 29 known genes. While the combined treatment induced a synergistic effect on the expression of LCK proto-oncogene and several genes related to protein synthesis/processing, a negative interaction effect was found for the expression of genes related to cytoskeleton and extracellular matrix assembly (SATB1 and Anexin III), cell cycle control (tyrosine kinase), and genes participating in DNA/RNA synthesis and repair (RNA helicase, FLAP endonuclease-1, DNA-3 glycosylase methyladenine, splicing factor SC35 and Soh1). The present data open the possibility to investigate the direct participation of specific genes, or gene products acting in concert in the mechanism underlying the cell response to radiation-induced DNA damage under the influence of PARP inhibitor.
Asunto(s)
Células 3T3/efectos de la radiación , Benzamidas/farmacología , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de la radiación , Ribonucleoproteínas , Células 3T3/efectos de los fármacos , Células 3T3/fisiología , Animales , Anexina A3/efectos de los fármacos , Anexina A3/genética , Anexina A3/efectos de la radiación , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Endodesoxirribonucleasas/efectos de los fármacos , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/efectos de la radiación , Endonucleasas de ADN Solapado , Rayos gamma , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/efectos de la radiación , Proteínas de Unión a la Región de Fijación a la Matriz/efectos de los fármacos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/efectos de la radiación , Ratones , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/efectos de la radiación , Análisis de Secuencia por Matrices de Oligonucleótidos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proto-Oncogenes Mas , ARN/biosíntesis , ARN/efectos de los fármacos , ARN/efectos de la radiación , ARN Helicasas/efectos de los fármacos , ARN Helicasas/genética , ARN Helicasas/efectos de la radiación , Factores de Empalme Serina-ArgininaRESUMEN
Prodigiosin (Prod, 1) is the parent member of a class of polypyrrole natural products that exhibit promising immunosuppressive and cytotoxic activity. They can facilitate copper-promoted oxidative double-strand (ds) DNA cleavage through reductive activation of Cu(II). This is triggered by oxidation of the electron-rich Prod molecule and may provide a basis for the cytotoxicity of the prodigiosins. To gain an understanding of this activity, we prepared several Prod analogues with various A-ring systems to examine their electrochemical properties in acetonitrile (MeCN) as a means to establish a basis for structure-reactivity relationships in copper-promoted nuclease activity. The intact bipyrrole (BP) chromophore is critical for the copper-mediated nuclease properties of the Prods. In fact, simple BP systems are shown to facilitate oxidative single-strand (ss) DNA cleavage. Replacement of the Prod A-pyrrole ring with alternative arenes (phenyl, furan-2-yl, or thiophen-2-yl) inhibits DNA strand scission and raises the half-peak oxidation potential (E(p/2)) of the Prod free base [E(p/2) = 0.44 V vs saturated calomel electrode (SCE) in MeCN] by ca. 200 mV. The same effect was achieved through attachment of an electron-withdrawing group (acetyl) at the 5'-position of the A-pyrrole ring. The structural modifications that inhibit DNA cleavage correlate with known structure-reactivity relationships of Prods against leukemia and melanoma cancer cells. The implications of our findings with regard to the cytotoxicity of the Prods are discussed.
Asunto(s)
Antibacterianos/síntesis química , Prodigiosina/análogos & derivados , Pirroles/síntesis química , Antibacterianos/química , Cobre/farmacología , Electroquímica , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/efectos de los fármacos , Oxidación-Reducción , Prodigiosina/síntesis química , Pirroles/química , Relación Estructura-ActividadRESUMEN
Cytolethal distending toxins (CDTs) block proliferation of mammalian cells by activating DNA damage-induced checkpoint responses. We demonstrate that the Haemophilus ducreyi CDT (HdCDT) induces phosphorylation of the histone H2AX as early as 1 h after intoxication and re-localization of the DNA repair complex Mre11 in HeLa cells with kinetics similar to those observed upon ionizing radiation. Early phosphorylation of H2AX was dependent on a functional Ataxia Telangiectasia mutated (ATM) kinase. Microinjection of a His-tagged HdCdtB subunit, homologous to the mammalian DNase I, was sufficient to induce re-localization of the Mre11 complex 1 h post treatment. However, the enzymatic potency was much lower than that exerted by bovine DNase I, which caused marked chromatin changes at 106 times lower concentrations than HdCdtB. H2AX phosphorylation and Mre11 re-localization were induced also in HdCDT-treated, non-proliferating dendritic cells (DCs) in a differentiation dependent manner, and resulted in cell death. The data highlight several novel aspects of CDTs biology. We demonstrate that the toxin activates DNA damage-associated molecules in an ATM-dependent manner, both in proliferating and non-proliferating cells, acting as other DNA damaging agents. Induction of apoptotic death of immature DCs by HdCDT may represent a previously unknown mechanism of immune evasion by CDT-producing microbes.
Asunto(s)
Toxinas Bacterianas/farmacología , Daño del ADN , Haemophilus ducreyi/patogenicidad , Proteínas de Saccharomyces cerevisiae , Proteínas de la Ataxia Telangiectasia Mutada , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Proteínas de Ciclo Celular , División Celular , Línea Celular , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Desoxirribonucleasa I/farmacología , Endodesoxirribonucleasas/efectos de los fármacos , Endodesoxirribonucleasas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Exodesoxirribonucleasas/efectos de los fármacos , Exodesoxirribonucleasas/metabolismo , Haemophilus ducreyi/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Terapia de Inmunosupresión , Fosfatidilinositol 3-Quinasas/farmacología , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/farmacología , Proteínas Recombinantes/farmacología , Factores de Tiempo , Proteínas Supresoras de TumorRESUMEN
Extracts of the human glioma cell line A1235 (lacking O(6)-methylguanine-DNA methyltransferase) are known to restore a G:T mismatch to a normal G:C pair in a G:T-containing model (45 bp) DNA substrate. Herein we demonstrate that substitution of G:T with O(6)-methylguanine:T (m6G:T) results in extract-induced intra-strand incision in the DNA at an efficiency comparable to that of complete repair of the G:T-containing substrate, although the m6G:T mispair serves as a poor substrate for later repair steps (e.g. gap filling, as judged by defective DNA repair synthesis). The A1235 extract, when supplemented with ATP and the four normal dNTPs, incises 5' to the mismatched T, as inferred by the generation of a single-stranded 20mer fragment. Unlike its parental (A1235) counterpart, an extract of the alkylation-tolerant derivative cell line A1235-MR4 produces no 20mer fragment, even when thymine-DNA glycosylase (TDG) is added to the reaction mixture. In contrast, the A1235 extract, when augmented with TDG, catalyzes enhanced incision at m6G:T in the 45 bp DNA, yielding 5-10-fold greater 20mer than that of either extract or TDG alone. Interestingly, the absence of m6G:T incision activity in the A1235-MR4 extract is similar to that seen for extracts of several known mismatch repair-deficient cell lines of colon tumor origin. Together these results suggest that derivative A1235-MR4 cells are defective in m6G:T incision activity and that the efficiency of this activity in the parental (A1235) cells may depend on the presence of several ill-defined mismatch repair recognition proteins along with TDG and ATP.
Asunto(s)
Disparidad de Par Base/genética , Reparación del ADN , ADN/metabolismo , Adenosina Trifosfato/farmacología , Secuencia de Bases , Extractos Celulares , Sistema Libre de Células/efectos de los fármacos , Sistema Libre de Células/metabolismo , ADN/química , ADN/genética , Desoxirribonucleasa (Dímero de Pirimidina) , Relación Dosis-Respuesta a Droga , Endodesoxirribonucleasas/efectos de los fármacos , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/farmacología , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Células HT29 , Humanos , Mutación , Nucleótidos/farmacología , Especificidad por Sustrato , Timina/química , Timina/metabolismo , Células Tumorales CultivadasRESUMEN
Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.
Asunto(s)
Desoxirribonucleasa (Dímero de Pirimidina) , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Thermoproteaceae/enzimología , Secuencia de Aminoácidos , Liasas de Carbono-Oxígeno/metabolismo , ADN Glicosilasas , ADN Recombinante/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Relación Dosis-Respuesta a Droga , Endodesoxirribonucleasas/efectos de los fármacos , Endodesoxirribonucleasas/genética , Estabilidad de Enzimas , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Filogenia , Homología de Secuencia de Aminoácido , Cloruro de Sodio/farmacología , Especificidad por Sustrato , TemperaturaRESUMEN
Subacute intoxication was induced by the oral administration of sodium nitrate 200 mg/kg during 150 days to Wistar rats. After the time had been up severe damaging were found in liver, kidney, heart and thymic tissues. In the liver cells the DNA fragmentation in "scale" manner was found, but not in kidney and heart cells. Simultaneously, the Ca2+, Mg(2+)-depended endonucleases activity were increased in the liver nuclei extracts under intoxication. It was suggested that increasing of apoptosis in liver is the universal reaction to toxins.
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Apoptosis/efectos de los fármacos , Carcinógenos/farmacología , Nitratos/envenenamiento , Enfermedad Aguda , Animales , ADN/efectos de los fármacos , Endodesoxirribonucleasas/efectos de los fármacos , Femenino , Masculino , Intoxicación/enzimología , Intoxicación/etiología , Intoxicación/patología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Neuronal survival is intricately linked to the maintenance of intact DNA. In contrast, neuronal degeneration following nitric oxide (NO) exposure is dependent, in part, on the degradation of DNA through programmed cell death (PCD). We therefore investigated in primary rat hippocampal neurons the role of endogenous deoxyribonucleases, enzymes responsible for metabolically derived DNA cleavage, during NO-induced neurodegeneration. Twenty-four hours following exposure to the NO generators sodium nitroprusside (300 microM) and SIN-1 (300 microM), neuronal survival was reduced from approximately 88 to 23%. Treatment with aurintricarboxylic acid (1-100 microM), an endonuclease inhibitor, during NO exposure increased neuronal survival from 23 to 80% and decreased DNA fragmentation from 70 to 30% over a 24-h period. Enhancement of endonuclease activity alone with zinc chelation actively decreased neuronal survival from approximately 80% to approximately 34%. DNA digestion assays identified not only two constitutively active endonucleases, an acidic endonuclease (pH 4.0-7.0) and a calcium/magnesium-dependent endonuclease (pH 7.2-8.0), but also a NO-inducible magnesium-dependent endonuclease (pH 8.0). In the absence of endonuclease activity, DNA degradation did not occur during NO application, suggesting that endonuclease activity was a requisite pathway for NO-induced PCD. In addition, NO independently altered intracellular pH in ranges that were physiologically relevant for the activity of the endonucleases responsible for DNA degradation. Our identification and characterization of specific neuronal endonucleases suggest that the constitutive endonucleases may play a role in the initial stages of NO-induced PCD, but the subsequent "downstream" degradation of DNA may ultimately be dependent upon the NO-inducible endonuclease.
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Apoptosis , Endodesoxirribonucleasas/biosíntesis , Neuronas/enzimología , Óxido Nítrico/metabolismo , Animales , Ácido Aurintricarboxílico/farmacología , Células Cultivadas , Endodesoxirribonucleasas/efectos de los fármacos , Inducción Enzimática , Etilenodiaminas/farmacología , Hipocampo/citología , Concentración de Iones de Hidrógeno , Líquido Intracelular , Molsidomina/análogos & derivados , Molsidomina/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Nitroprusiato/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
An endonuclease named DNase gamma was purified to apparent homogeneity from rat splenocyte nuclei and its properties were characterized. We also determined the NH2-terminal and partial amino acid sequences of the proteolytic internal peptides. The molecular mass of gamma DNase was 33,000 daltons as determined by SDS-polyacrylamide gel electrophoresis. A native molecular mass of 30,000 was estimated by gel filtration. Purified DNase gamma is active in the presence of both Ca2+ and Mg2+ or Mn2+ alone and inhibited by Co2+, Ni2+, Cu2+, and especially Zn2+. Maximal activity was achieved at pH 7.2 in Mops-NaOH buffer. The sequence data on the NH2-terminal and seven internal peptides obtained by sequential digestions with Achromobacter protease I and endoproteinase Asp-N revealed that DNase gamma is a novel endonuclease that shows sequence homology with DNase I.
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Endodesoxirribonucleasas/química , Bazo/enzimología , Secuencia de Aminoácidos , Animales , Calcio/farmacología , Cationes Bivalentes/farmacología , Cromatografía Líquida de Alta Presión , Desoxirribonucleasa I/química , Desoxirribonucleasa I/clasificación , Endodesoxirribonucleasas/clasificación , Endodesoxirribonucleasas/efectos de los fármacos , Endodesoxirribonucleasas/metabolismo , Magnesio/farmacología , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Análisis de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
One approach to discriminate among specific DNases in apoptosis is to use inhibitors specific for each endonuclease. Zn2+ is known to inhibit Ca(2+)- and Mg(2+)-dependent endonuclease enzymatic activities during apoptosis. Acidic DNases were thought to be insensitive to Zn2+. In this paper, we analyse the effects of Zn2+ on activity of DNase II, either purified or in nuclei from lens fiber cells. These cells follow a physiological nuclear degeneration with DNase II accumulation in their nuclei. We show that Zn2+ is able to inhibit also this acidic endonuclease at a concentration of 1-6 mM. At a higher concentration of Zn2+, DNA is extensively degraded during the assay, masking the inhibition of the enzyme. This DNA degradation in the presence of Zn2+ has led to an overestimation of the activity of DNase II in studies of apoptosis. Hence, Zn2+ cannot be used to specifically identify one endonuclease among the different DNases involved in nuclear degradation during programmed cell death.
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Apoptosis , Endodesoxirribonucleasas/efectos de los fármacos , Endodesoxirribonucleasas/metabolismo , Zinc/farmacología , Animales , Apoptosis/efectos de los fármacos , Bovinos , Embrión de Pollo , Fragmentación del ADN/efectos de los fármacos , Endodesoxirribonucleasas/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Escherichia coli , Corteza del Cristalino/citología , Corteza del Cristalino/efectos de los fármacos , Corteza del Cristalino/enzimología , PorcinosRESUMEN
The E. coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair. Using recombination intermediates made by RecA protein, we have identified specific "hotspots" for RuvC resolution. Characterization of these sites reveals a common tetranucleotide sequence, with the consensus 5'-A/TTT decreases G/C-3'. The correct orientation of the resolution site is required for cleavage. These observations suggest that the strand bias of this sequence will affect the outcome of recombinational crosses by directing resolution to either "patch" or "splice" recombinant products. Mutation of the consensus site in synthetic Holliday junctions abolishes or significantly reduces the efficiency of cleavage, although binding is unaffected, demonstrating that junction recognition and incision are biochemically separable events. We propose that efficient RuvC resolution requires the translocation of Holliday junctions to specific cleavage sites, thus providing a biochemical basis for the similar genetic defects observed in ruvA, ruvB, and ruvC mutants.