RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged beta-coronavirus that enter cells via two routes, direct fusion at the plasma membrane or endocytosis followed by fusion with the late endosome/lysosome. While the viral receptor, ACE2, multiple entry factors and the mechanism of fusion of the virus at the plasma membrane have been investigated extensively, viral entry via the endocytic pathway is less understood. By using a human hepatocarcinoma cell line, Huh-7, which is resistant to the antiviral action of the TMPRSS2 inhibitor camostat, we discovered that SARS-CoV-2 entry is not dependent on dynamin but on cholesterol. ADP-ribosylation factor 6 (ARF6) has been described as a host factor for SARS-CoV-2 replication and is involved in the entry and infection of several pathogenic viruses. Using CRISPR/Cas9 genetic deletion, a modest reduction in SARS-CoV-2 uptake and infection in Huh-7 was observed. In addition, pharmacological inhibition of ARF6 with the small molecule NAV-2729 showed a dose-dependent reduction of viral infection. Importantly, NAV-2729 also reduced SARS-CoV-2 viral loads in more physiological models of infection: Calu-3 cells and kidney organoids. This highlighted a role for ARF6 in multiple cell contexts. Together, these experiments point to ARF6 as a putative target to develop antiviral strategies against SARS-CoV-2.
Assuntos
COVID-19 , Humanos , Fator 6 de Ribosilação do ADP , Antivirais/farmacologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do VírusRESUMO
SARS-CoV-2 is a newly emerged beta-coronavirus that enter cells via two routes, direct fusion at the plasma membrane or endocytosis followed by fusion with the late endosome/lysosome. While the viral receptor, ACE2, multiple entry factors, and the mechanism of fusion of the virus at the plasma membrane have been extensively investigated, viral entry via the endocytic pathway is less understood. By using a human hepatocarcinoma cell line, Huh-7, which is resistant to the antiviral action of the TMPRSS2 inhibitor camostat, we discovered that SARS-CoV-2 entry is not dependent on dynamin but dependent on cholesterol. ADP-ribosylation factor 6 (ARF6) has been described as a host factor for SARS-CoV-2 replication and it is involved in the entry and infection of several pathogenic viruses. Using CRISPR-Cas9 genetic deletion, we observed that ARF6 is important for SARS-CoV-2 uptake and infection in Huh-7. This finding was corroborated using a pharmacologic inhibitor, whereby the ARF6 inhibitor NAV-2729 showed a dose-dependent inhibition of viral infection. Importantly, NAV-2729 reduced SARS-CoV-2 viral loads also in more physiologic models of infection: Calu-3 and kidney organoids. This highlighted the importance of ARF6 in multiple cell contexts. Together, these experiments points to ARF6 as a putative target to develop antiviral strategies against SARS-CoV-2.
RESUMO
OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.
Assuntos
Apoptose , Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/genética , Neoplasias Hepáticas/virologia , Transativadores/genética , Replicação Viral , Adulto , Idoso , DNA Viral/genética , Feminino , Genótipo , Células Hep G2 , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/virologia , Humanos , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Homologia Estrutural de Proteína , Transativadores/química , Proteínas Virais Reguladoras e AcessóriasRESUMO
Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed the NS5B polymerase genetic variability in circulating HCV genotypes/subtypes and its impact on the genetic barrier for the development of resistance to clinically relevant nucleoside inhibitors (NIs)/nonnucleoside inhibitors (NNIs). The study included 1,145 NS5B polymerase sequences retrieved from the Los Alamos HCV database and GenBank. The genetic barrier was calculated for drug resistance emergence. Prevalence and genetic barrier were calculated for 1 major NI and 32 NNI resistance variants (13 major and 19 minor) at 21 total NS5B positions. Docking calculations were used to analyze sofosbuvir affinity toward the diverse HCV genotypes. Overall, NS5B polymerase was moderately conserved among all HCV genotypes, with 313/591 amino acid residues (53.0%) showing ≤1% variability and 83/591 residues (14.0%) showing high variability (≥25.1%). Nine NNI resistance variants (2 major variants, 414L and 423I; 7 minor variants, 316N, 421V, 445F, 482L, 494A, 499A, and 556G) were found as natural polymorphisms in selected genotypes. In particular, 414L and 423I were found in HCV genotype 4 (HCV-4) (n = 14/38, 36.8%) and in all HCV-5 sequences (n = 17, 100%), respectively. Regardless of HCV genotype, the 282T major NI resistance variant and 10 major NNI resistance variants (316Y, 414L, 423I/T/V, 448H, 486V, 495L, 554D, and 559G) always required a single nucleotide substitution to be generated. Conversely, the other 3 major NNI resistance variants (414T, 419S, and 422K) were associated with a different genetic barrier score development among the six HCV genotypes. Sofosbuvir docking analysis highlighted a better ligand affinity toward HCV-2 than toward HCV-3, in agreement with the experimental observations. The genetic variability among HCV genotypes, particularly with the presence of polymorphisms at NNI resistance positions, could affect their responsiveness to NS5B inhibitors. A pretherapy HCV NS5B sequencing could help to provide patients with the full efficacy of NNI-containing regimens.
Assuntos
Hepacivirus/genética , Antivirais/farmacologia , Genótipo , Hepacivirus/efeitos dos fármacos , Mutação , Polimorfismo Genético/genética , Estrutura Secundária de Proteína , Sofosbuvir , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Leukocytes participate in different ways in inflammatory diseases. The nature of the disease can be seen as a reflection, in part, of the migration patterns of different leukocyte types in asthma versus inflammatory bowel disease (IBD). The molecular basis that underlies the selective recruitment of distinct leukocytes to unique microenvironments is a result of the interplay of selectins, integrins and chemoattractants. The ability to understand and dissect these pathways in disease processes will be an important tool for the development of novel therapeutics with highly effective suppressive potential and greatly reduced side-effects.
RESUMO
Antisense oligonucleotides have the ability to inhibit gene expression in viral infections, malignancy, and other diseases. Even though much work has been accomplished with oligonucleotides demonstrating in vitro therapeutic effects, little work has been done to address how these molecules gain access to the cell. One of the plausible means of entrance could be through passive diffusion of the oligonucleotides through the cellular lipid bilayer. To enhance membrane permeability of oligonucleotides lipophilic moieties at the 2' position of the ribose ring have been added. To evaluate the effect of this modification, a liposome system was used. The oligonucleotides evaluated were a series composed of poly A 10mers phosphorothioates labeled at the 5' end with fluorescein and modified at the 2' position of the ribose ring with lipophilic alkyl chains ranging from methyl to nonyl. Efflux studies were accomplished by monitoring the appearance of the oligonucleotide in the incubation medium. There were modest but significant differences between the efflux half-life times of the 2'-modified compounds and the control compound. The values ranged from approximately 6 days for the control, unmodified compound to 4.6 days for the propyl modification. The nonyl derivative had a longer efflux half-life time (8.3 days) compared with the control, unmodified phosphorothioate oligonucleotide.
Assuntos
Oligonucleotídeos Antissenso/química , Tionucleotídeos/química , Fenômenos Químicos , Físico-Química , Difusão , Meia-Vida , Bicamadas Lipídicas , Lipossomos , Lipídeos de Membrana/química , PermeabilidadeRESUMO
Papillomaviruses induce benign proliferative lesions, such as genital warts, in humans. The E2 gene product is thought to play a major role in the regulation of viral transcription and DNA replication and may represent a rational target for an antisense oligonucleotide drug action. Phosphorothioate oligonucleotides complementary to E2 mRNAs were synthesized and tested in a series of in vitro bovine papillomavirus (BPV) and human papillomavirus (HPV) models for the ability to inhibit E2 transactivation and virus-induced focus formation. The most active BPV-specific compounds were complementary to the mRNA cap region (ISIS 1751), the translation initiation region for the full-length E2 transactivator (ISIS 1753), and the translation initiation region for the E2 transrepressor mRNA (ISIS 1755). ISIS 1751 and ISIS 1753 were found to reduce E2-dependent transactivation and viral focus formation in a sequence-specific and concentration-dependent manner. ISIS 1755 increased E2 transactivation in a dose-dependent manner but had no effect on focus formation. Oligonucleotides with a chain length of 20 residues had optimal activity in the E2 transactivation assay. On the basis of the above observations, ISIS 2105, a 20-residue phosphorothioate oligonucleotide targeted to the translation initiation of both HPV type 6 (HPV-6) and HPV-11 E2 mRNA, was designed and shown to inhibit E2-dependent transactivation by HPV-11 E2 expressed from a surrogate promoter. These observations support the rationale of E2 as a target for antiviral therapy against papillomavirus infections and specifically identify ISIS 2105 as a candidate antisense oligonucleotide for the treatment of genital warts induced by HPV-6 and HPV-11.
Assuntos
Antivirais/uso terapêutico , Condiloma Acuminado/tratamento farmacológico , Proteínas de Ligação a DNA/genética , Papillomaviridae/genética , RNA Antissenso/genética , RNA Mensageiro/genética , Tionucleotídeos/uso terapêutico , Proteínas Virais/genética , Animais , Sequência de Bases , Papillomavirus Bovino 1/efeitos dos fármacos , Papillomavirus Bovino 1/genética , Condiloma Acuminado/genética , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Papillomaviridae/efeitos dos fármacos , Capuzes de RNA/metabolismo , RNA Viral/genética , Ribonuclease H/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
The human nucleolar antigen p120 was detected with an anti-p120 monoclonal antibody (MAbp120) in most human malignant tumors (Freeman et al., Cancer Research, 48, 1244-1251, 1988). Stable transfection of the sense p120 cDNA caused malignant transformation of NIH/3T3 cells in vitro, and the antisense p120 constructs markedly delayed the growth of these transformed cells (Perlaky et al., Cancer Research, 52, 428-436, 1992). Several p120 antisense phosphorothioate oligonucleotides designed to hybridize with different regions of the p120 sequence were screened on human tumor cell lines in vitro. Marked growth inhibition of HeLa, LOX and HRCC cell lines was found, particularly with antisense p120 oligonucleotide ISIS 3466 in combination with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); oligonucleotide ISIS 3466 is complementary to a non-translated region at the 3' end of the molecule. Preliminary in vivo studies on human LOX ascites tumor in nude mice showed marked inhibitory effects on tumor growth by the antisense oligonucleotide ISIS 3466 in the presence of DOTMA when treated on alternate days.
Assuntos
Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Oligonucleotídeos Antissenso/farmacologia , Animais , Sequência de Bases , Ensaios de Seleção de Medicamentos Antitumorais , Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proteínas de Neoplasias/efeitos dos fármacos , Transplante de Neoplasias , Proteínas Nucleares/efeitos dos fármacos , Proteínas Metiltransferases , Células Tumorais Cultivadas/efeitos dos fármacos , tRNA MetiltransferasesRESUMO
We have investigated the use of a cationic lipid preparation to enhance antisense oligonucleotide activity in human umbilical vein endothelial cells. A liposomal preparation containing the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) was found to increase by at least 1000-fold the potency of an antisense oligonucleotide (ISIS 1570) that hybridizes to the AUG translation initiation codon of human intercellular adhesion molecule-1. In the presence of 8 microM DOTMA, 6-15-fold more 35S-ISIS 1570 associated with cells, at oligonucleotide concentrations from 0.01 to 5 microM, than did in the absence of DOTMA. Both 35S-ISIS 1570 association with cells and antisense activity were increased as a function of DOTMA concentration and with increasing time of incubation with the cationic lipid. Fluorescein-labeled ISIS 1570 was used to assess the intracellular distribution of the oligonucleotide in the presence and absence of DOTMA. In the absence of DOTMA, the oligonucleotide localized to discrete structures in the cytoplasm of the cell, resulting in a punctate fluorescence pattern. In the presence of DOTMA, cellular fluorescence markedly increased and the oligonucleotide localized within the nucleus, as well as to discrete structures in the cytoplasm. Accumulation of the oligonucleotide in the nucleus in the presence of DOTMA was time and temperature dependent. Nuclear accumulation was inhibited by preincubation of the cells with monensin but not chloroquine, NH4Cl, nocodazole, colcemid, or brefeldin A. These data demonstrate that cationic lipids increase antisense activity by increasing the amount of oligonucleotide associated with cells and altering intracellular distribution of the oligonucleotide.
Assuntos
Oligonucleotídeos Antissenso/farmacocinética , Compostos de Amônio Quaternário/farmacologia , Tionucleotídeos/farmacocinética , Sequência de Bases , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular , Cinética , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Frações Subcelulares/metabolismo , Tionucleotídeos/farmacologiaRESUMO
Human cells contain two topoisomerase II isozymes named topo II alpha and topo II beta. The complementary DNAs for both enzymes have been cloned. The topo II alpha and topo II beta complementary DNAs hybridized to unique sequences of human, rodent, and chicken DNAs in Southern blots. The human topo II alpha gene has previously been mapped to chromosome 17. We confirmed the chromosomal location of topo II alpha and mapped the topo II beta gene to chromosome 3. In addition, topo II beta exhibits genetic polymorphism as has been reported for topoisomerases I and II alpha.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 17/enzimologia , Cromossomos Humanos Par 3/enzimologia , DNA Topoisomerases Tipo II/genética , Isoenzimas/genética , Southern Blotting , HumanosRESUMO
The use of antisense oligonucleotide as pharmacologic agents is a derivative of the central dogma of molecular biology and knowledge of the physical and chemical properties that govern the structure of nucleic acids. Oligonucleotides have been reported to inhibit the growth of a large number of viruses in cell culture, as well as the expression of numerous oncogenes, a variety of normal genes and transfected reporter genes controlled by several regulatory elements. The therapeutic activity of antisense compounds in animal disease models have also been reported. This review provides some general conclusions and trends regarding the pharmacologic action of antisense oligonucleotides, that can be formulated from studies previously reported in the literature. In addition, data is highlighted for two specific examples in which antisense oligonucleotides have demonstrated activity against herpes viruses and intracellular adhesion molecule RNA targets.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Animais , Moléculas de Adesão Celular/efeitos dos fármacos , Desenho de Fármacos , Humanos , Simplexvirus/efeitos dos fármacosRESUMO
The cellular content of 170kD and 180kD topoisomerase II was studied as a function of the proliferation state and cell cycle position in NIH-3T3 cells. When the cells were synchronized by serum starvation and then stimulated to enter the cell cycle by addition of fresh growth medium, the amount of 170kD topoisomerase II present was undetectable until the cells reached late S phase, peaked in G2-M phase cells, and decreased as the cells completed mitosis. The amount of 180kD topoisomerase II was constant once the cells entered the cell cycle. When exponentially growing cells were induced to enter G0 by serum starvation, the amount of 170kD topoisomerase II decreased in parallel with the loss of cells from the S and G2-M phases of the cell cycle and was undetectable once all of the cells reached G0. In contrast, the 180kD enzyme was still present after all of the cells had entered G0. The tightness of association of the two enzymes with chromatin was measured by determining the concentration of salt required to extract them from isolated nuclei. The 180kD enzyme required a higher concentration of NaCl for extraction than did the 170kD enzyme. The different patterns of expression of the two forms of topoisomerase II suggest that they perform different functions in cells.
Assuntos
Ciclo Celular , DNA Topoisomerases Tipo II/biossíntese , Fibroblastos/enzimologia , Animais , Divisão Celular , Células Cultivadas , Cromatina/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/fisiologia , Indução Enzimática , Fibroblastos/citologia , Camundongos , Peso MolecularRESUMO
Incubation of cultured rat aortic smooth muscle cells (A-10) with activators of cyclic nucleotides resulted in transiently increased activity of extractable topoisomerase I or topoisomerase II. ANF, which induces cGMP accumulation, potentiated camptothecin-induced, topoisomerase I linked DNA strand breakage and increased the specific activity of extractable topoisomerase I (maximum activity 5-15 min after treatment), but had no effect on topoisomerase II activity. These effects are similar to those reported for AVP and phorbol esters, activators of protein kinase C. Forskolin and isoproterenol, which induce cAMP accumulation, activated extractable topoisomerase II (maximum 5-15 min after treatment), but not topoisomerase I. Permeable cyclic nucleotide analogs dBcAMP and 8BrcGMP selectively activated extractable topoisomerase II and topoisomerase I activities, respectively. Activation of topoisomerase I by either AVP or PdBu was attenuated by cotreatment with 8BrcGMP or dBcAMP, and activation of topoisomerase II by dBcAMP was attenuated by cotreatment with AVP or PdBU, suggesting that elements of the protein kinase C and the cyclic nucleotide linked signal-transduction pathways can interact to modify nuclear enzymic activity. IBMX, which elevates intracellular cAMP and cGMP, increased the extractable activities of both topoisomerase I and topoisomerase II. Thus, topoisomerase activity in cells may be governed in part by cyclic nucleotide levels.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Fosfolipídeos/fisiologia , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Extratos Celulares/isolamento & purificação , Núcleo Celular/enzimologia , Células Cultivadas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Dano ao DNA , Ativação Enzimática/efeitos dos fármacos , Proteína Quinase C/efeitos dos fármacosRESUMO
Spirogermanium (1; 8,8-diethyl-N,N-dimethyl-2-aza-8- germaspiro[4.5]decane-2-propanamine dihydrochloride) is a potent cytotoxic agent in vitro which has demonstrated limited activity in experimental animal tumor models. Subsequently, it has been reported that spirogermanium has antiarthritic and suppressor cell-inducing activity. We have synthesized a series of substituted 8-hetero-2-azaspiro[4.5]decane and 9-hetero-3-azaspiro[5.5]undecane analogues of spirogermanium to identify the heteroatom requirements for in vivo antiarthritic and suppressor cell-inducing activity. This structure-activity relationship study has identified that appropriately substituted silicon and carbon analogues of spirogermanium retain both antiarthritic and immunosuppressive activity, with the 8,8-dipropyl (carbon) analogue being among the most active. Following the identification of N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine++ + dihydrochloride (9) as a more active analogue than spirogermanium, a series of 8,8-dipropyl analogues with various amine substituents were synthesized. A number of these analogues had activity similar to that of 9. A correlation between activity in the adjuvant arthritic rat and the ability to induce suppressor cells (r = 0.894, p less than 0.001) suggests an association between the two pharmacologic effects. While the precise biochemical mechanism(s) for the pharmacological activity is unclear, these data suggest that compounds within this series, e.g., N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine++ + dihydrochloride, may provide effective therapy in diseases of autoimmune origin and/or the prevention of rejection in tissue transplantation.
Assuntos
Artrite Experimental/tratamento farmacológico , Compostos Aza/farmacologia , Imunossupressores/farmacologia , Compostos de Espiro/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Compostos Aza/síntese química , Compostos Aza/uso terapêutico , Fenômenos Químicos , Química , Imunossupressores/síntese química , Imunossupressores/uso terapêutico , Masculino , Estrutura Molecular , Compostos Organometálicos/farmacologia , Compostos Organometálicos/uso terapêutico , Ratos , Ratos Endogâmicos Lew , Compostos de Espiro/síntese química , Compostos de Espiro/uso terapêutico , Relação Estrutura-Atividade , Linfócitos T Reguladores/imunologiaRESUMO
The efflux of gold from red blood cells (RBCs) exposed to 10-100 microM auranofin [the second generation chrysotherapy agent, triethylphosphine-(2,3,4,6-tetra-O-acetyl-1-beta-D-glucopyranosato -S-)gold(I)] was studied. RBCs in whole blood were allowed to accumulate gold, and then were placed in fresh plasma or buffered saline solution. In plasma, the kinetics of efflux were first order in gold with an apparent rate constant of 0.81 +/- 0.18 hr-1. Serum albumin, in plasma or added to a buffered solution, shifted the equilibrium between intra- and extracellular gold in favor of the latter (compared to saline solution). [14C]Glutathione, generated by in situ labeling, also effluxed and associated with the albumin and gold, providing the first direct evidence that the albumin-gold-glutathione complex (AlbSAuSG) may be a circulating metabolite of auranofin formed after both of the original ligands of auranofin are displaced.
Assuntos
Albuminas/metabolismo , Auranofina/metabolismo , Eritrócitos/metabolismo , Glutationa/metabolismo , Ouro/metabolismo , Auranofina/farmacologia , Sítios de Ligação , Células Cultivadas , Cromatografia em Gel , Humanos , CinéticaRESUMO
SK&F 105685 (N,N-Dimethyl-8,8-dipropyl-2-azaspiro[4,5]decane-2- propanamine dihydrochloride) is a novel azaspirane which has therapeutic activity in rat models of autoimmune disease. In this study, we have demonstrated that SK&F 105685 is a potent inducer of non-specific suppressor cells (SC). Oral administration of 15-30 mg/kg/day results in the generation of SC in the spleen, lymph nodes and bone marrow, but not the thymus of Lewis rats. Splenic SC suppress Con-A-induced proliferation in co-culture assays at effector-responder ratios of 1:1 to 1:64. SC are radiation resistant (2000 R), non-T, non-B cells, partially adherent to plastic surfaces and are enriched in a 1.07 g/ml fraction of a Percoll density gradient. Their activity is increased, rather than ablated, by indomethacin. No definitive changes in Ig+, OX-19+, OX-8+, W3/25+ or asialo GM1+ cells could be detected in the spleens of treated rats compared to control untreated animals. Elevated levels of both radiation-sensitive and radiation-resistant suppressor cells were found in the bone marrow of treated rats in addition to the radiosensitive SC normally present in this tissue.
Assuntos
Compostos de Espiro/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Adjuvantes Imunológicos/farmacologia , Administração Oral , Animais , Separação Celular , Ativação Linfocitária/efeitos dos fármacos , Tecido Linfoide/citologia , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/imunologia , Masculino , Tolerância a Radiação , Ratos , Ratos Endogâmicos Lew , Compostos de Espiro/administração & dosagem , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos da radiaçãoRESUMO
The ATP-dependent unknotting of phage P4 DNA is a highly specific assay for type II topoisomerases. Despite the unique specificity of the assay, however, its semiquantitative design has limited its use in studying the biochemical properties of these enzymes. To overcome this problem, we have modified the P4 DNA unknotting assay to provide a sensitive and reproducible method for quantifying topoisomerase II activity. Methods are described for accurate measurement of 10-100 ng of unknotted P4 DNA. Under the assay conditions employed, the initial rate of topoisomerase II activity was linear through 30 min. The quantitative assay has been used to determine biochemical and pharmacological parameters of purified topoisomerase II (p170). No topoisomerase II activity was observed in the absence of ATP; enzymatic activity was optimal between 0.5 and 1.0 mM ATP, but substrate inhibition occurred at concentrations above 1 mM. Eadie-Hofstee analysis with varying ATP concentrations gave an apparent Km for ATP of 0.24 mM and a maximal velocity under these conditions of 7.4 ng P4 DNA unknotted/min/ng topoisomerase II. IC50 values were determined for several topoisomerase inhibitors, including amsacrine, teniposide, and novobiocin. Inhibition by teniposide was found to be uncompetitive versus ATP, with a Ki of 3.7 microM. In contrast, inhibition by novobiocin was competitive versus ATP, indicating that teniposide and novobiocin inhibit topoisomerase II by different mechanisms.
Assuntos
DNA Topoisomerases Tipo II/farmacologia , DNA Topoisomerases Tipo I/efeitos dos fármacos , Amsacrina/farmacologia , Bacteriófagos/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Etídio/farmacologia , Relação Estrutura-Atividade , Teniposídeo/farmacologia , Inibidores da Topoisomerase IIRESUMO
A combination of tumor necrosis factor (TNF) and the topoisomerase I inhibitor, camptothecin, or the topoisomerase II inhibitors, teniposide and amsacrine, produced dose-dependent synergistic cytotoxicity against the murine L929 fibrosarcoma cells. Similar synergy was not observed with a combination of TNF and bleomycin. To define the role of TNF in the augmentation of tumor cell killing by topoisomerase I or II inhibitors, the effect of TNF on the production of enzyme-linked DNA strand breaks induced in cells by topoisomerase inhibitors was investigated. L929 cells incubated for 1 h with the topoisomerase inhibitors contained protein-linked strand breaks. In contrast, TNF alone did not induce DNA strand breakage. However, when cells were incubated simultaneously with TNF and camptothecin, amsacrine, Adriamycin, actinomycin D, teniposide, or etoposide, increased numbers of strand breaks were produced. Preincubation of the cells with TNF for 30 min or 3 h before the addition of camptothecin or etoposide resulted in no more strand breaks than that observed in cells incubated with the drugs alone. TNF treatment of L929 cells produced a rapid and transient increase in specific activity of extractable topoisomerases I and II. These increases were maximum at 2-5 min of TNF treatment and by 30 min the activities of extractable enzymes were equal to or less than those detected in extracts from untreated cell controls. The transient nature of the increase in extractable topoisomerase activity may explain the kinetics and significance of the order of addition of TNF and inhibitors for maximal synergistic activity. These data are consistent also with a role for topoisomerase-linked DNA lesions in the TNF-mediated potentiation of killing of L929 cells by topoisomerase inhibitors.
Assuntos
Dano ao DNA , Inibidores da Topoisomerase I , Inibidores da Topoisomerase II , Fator de Necrose Tumoral alfa/farmacologia , Amsacrina/farmacologia , Animais , Camptotecina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , DNA Topoisomerases Tipo I/análise , DNA Topoisomerases Tipo II/análise , Sinergismo Farmacológico , Camundongos , Células Tumorais CultivadasRESUMO
The activity of topoisomerase II and the cellular content of the 170kD and 180kD forms of the enzyme were studied as functions of transformation and growth state by using normal and ras-transformed NIH-3T3 cells. Total topoisomerase II activity, as measured by the unknotting of P4 DNA, was higher in ras-transformed than in normal cells in similar growth states, and was higher in exponentially growing than in plateau cells for both cell lines. Total topoisomerase II levels, as measured by immunoblotting, showed a similar dependence on transformation and growth state. The relative amounts of the 170kD and 180kD forms of the enzyme varied as a function of transformation and growth state. The proportion of 170kD topoisomerase II was higher in ras-transformed than in untransformed cells and depended much less on growth state in the ras-transformed cells. The topoisomerase II activity in extracts of ras-transformed cells was more sensitive to inhibition by teniposide and merbarone, drugs which selectively inhibit the 170kD form of topoisomerase II. The ras-transformed cells were also more sensitive to the cytotoxic effects of these drugs. An increase in the relative cellular content of 170kD topoisomerase II is characteristic of ras-transformed 3T3 cells, and the levels of this form of the enzyme appear to be less dependent on proliferation state than in untransformed cells. The susceptibility of certain tumors to killing by topoisomerase II-directed drugs may be due to a higher proportion of 170kD enzyme as well as a higher level of total topoisomerase II activity.
Assuntos
Transformação Celular Neoplásica/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Genes ras , Animais , Northern Blotting , Western Blotting , Divisão Celular , Linhagem Celular , Núcleo Celular/enzimologia , Dano ao DNA , DNA Topoisomerases Tipo II/classificação , Camundongos , Peso Molecular , Novobiocina/farmacologia , RNA Mensageiro/genética , Teniposídeo/farmacologia , Tiobarbitúricos/farmacologia , Inibidores da Topoisomerase IIRESUMO
We report the cytotoxicity toward B16 cells and antitumor activity in three transplantable tumor models of a series of ionic, tetrahedral, bischelated gold diphosphine complexes of the type [Au1(R2PYPR2')2]X, where Y = (CH2)2, (CH2)3, or cis-CH = CH. The anion (X = Cl, Br, I, CH3SO3, NO3, PF6) had little effect upon activity. The R = R' = phenyl complexes 1, 7, and 8 [Y = (CH2)2, (CH2)3, cis-CH = CH, X = Cl] were the most active against P388 leukemia, with an increase in lifespan ranging from 83 to 92% and were also active against M5076 sarcoma and B16 melanoma. Complexes with pyridyl or fluorophenyl substituents had reduced activities. For the latter, 19F and 31P NMR were used to verify the formation of bischelated gold(I) complexes in solution. The reduced activity of the complex with R = Et and R' = Ph and inactivity with R = R' = Et are discussed in terms of their increased reactivity as reducing agents. 31P NMR studies show that [AuI(Et2P(CH2)2PPh2)2]Cl readily reacts with serum, albumin, and Cu2+ ions to give oxidized ligand.