RESUMO
NF-κB signaling plays a pivotal role in control of the inflammatory response. We investigated how the dynamics and function of NF-κB were affected by temperature within the mammalian physiological range (34 °C to 40 °C). An increase in temperature led to an increase in NF-κB nuclear/cytoplasmic oscillation frequency following Tumor Necrosis Factor alpha (TNFα) stimulation. Mathematical modeling suggested that this temperature sensitivity might be due to an A20-dependent mechanism, and A20 silencing removed the sensitivity to increased temperature. The timing of the early response of a key set of NF-κB target genes showed strong temperature dependence. The cytokine-induced expression of many (but not all) later genes was insensitive to temperature change (suggesting that they might be functionally temperature-compensated). Moreover, a set of temperature- and TNFα-regulated genes were implicated in NF-κB cross-talk with key cell-fate-controlling pathways. In conclusion, NF-κB dynamics and target gene expression are modulated by temperature and can accurately transmit multidimensional information to control inflammation.
Assuntos
Regulação da Expressão Gênica/fisiologia , NF-kappa B/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/metabolismo , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Inflamação , Camundongos , NF-kappa B/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Temperatura , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/análise , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genéticaRESUMO
Interleukin-1ß (IL-1ß) is a critical regulator of the inflammatory response. IL-1ß is not secreted through the conventional ER-Golgi route of protein secretion, and to date its mechanism of release has been unknown. Crucially, its secretion depends upon the processing of a precursor form following the activation of the multimolecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1ß release process, in combination with biochemical, biophysical, and real-time single-cell confocal microscopy with macrophage cells expressing Venus-labelled IL-1ß, we have discovered that the secretion of IL-1ß after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus, in macrophages the release of IL-1ß in response to inflammasome activation appears to be a secretory process independent of nonspecific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1ß release is distinct from the unconventional secretory mechanism employed by its structural homologues fibroblast growth factor 2 (FGF2) or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into the mechanisms of protein release.
Assuntos
Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Caspase 1/metabolismo , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Taninos Hidrolisáveis/farmacologia , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , Lipossomos/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Microscopia de Fluorescência , Permeabilidade/efeitos dos fármacos , Potássio/análise , Potássio/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Live-cell imaging of fluorescent fusion proteins has transformed our understanding of mammalian cell signalling and function. However, some cellular systems such as immune cells are unsuitable or refractory to many existing transgene delivery methods thus limiting systematic analyses. Here, a flexible lentiviral gene transfer platform for dynamic time-lapse imaging has been developed and validated with single-molecule spectroscopy, mathematical modelling and transcriptomics and used for analysis of a set of inflammation-related signalling networks. Time-lapse imaging of nuclear factor kappa B (NF-κB), signal transducer and activator of transcription (STATs) and nuclear factor of activated T-cells (NFAT) in mammalian immune cell lines provided evidence for heterogeneous temporal encoding of inflammatory signals. In particular, the absolute quantification of single-cell responses over time via fluorescent correlation spectroscopy (FCS) showed that NF-κB p65 activation in response to tumour necrosis factor α (TNFα) was differentially encoded in variable amplitude of nuclear translocation between immune and non-immune cells. The absolute number of activated molecules was dictated in part by the cell size, suggesting a morphology-dependent regulatory mechanism. The developed platform will enable further absolute quantitative analyses of the dynamic interactions between signalling networks, in and between individual cells, allowing better integration with mathematical models of signalling networks.
Assuntos
Técnicas de Transferência de Genes , Sistema Imunitário/citologia , Sistema Imunitário/metabolismo , Lentivirus/genética , Imagem com Lapso de Tempo/métodos , Animais , Linhagem Celular , Células HEK293 , Humanos , Fenômenos do Sistema Imunitário/genética , Células Jurkat , Camundongos , Microscopia Confocal , Modelos Imunológicos , Fatores de Transcrição NFATC/genética , Células RAW 264.7 , Fatores de Transcrição STAT/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Análise de Célula Única/métodos , Fator de Transcrição RelA/genéticaRESUMO
The activation and regulation of target genes by the tumour-suppressor p53 dictates the fate of a cell, with cell cycle arrest or apoptosis being two distinct outcomes. PERP (p53 apoptosis effector related to PMP-22), a p53 transcriptional target, is induced specifically during apoptosis but not cell cycle arrest. Downregulation of PERP is associated with the aggressive, monosomy 3-type of uveal melanoma (UM), the most common primary intraocular tumour in adults, and increased PERP expression has a pro-apoptotic effect in UM cells. Here, we identify a novel effect of PERP expression, as elevated PERP protein positively influences active levels of its own transcriptional regulator, p53. Using fluorescent fusion proteins of PERP, p53 and MDM2, we demonstrate in single living UM cells that PERP expression significantly enhances p53 activity and its nuclear localization, increases p53-dependent transcription (including that of MDM2) while allowing oscillatory nucleo-cytoplasmic shuttling of p53/MDM2 complexes. Phosphorylation of p53 serine residues that interfere with the interaction between p53 and its negative regulator MDM2 and enhance pro-apoptotic gene transcription also occurs subsequent to PERP expression. These results implicate a role for PERP in amplifying functional p53 levels that promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Linhagem Celular Tumoral , Genes Supressores de Tumor , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/fisiopatologia , Proteínas de Membrana/genética , Fosforilação , Ligação Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/fisiopatologiaRESUMO
Medulloblastoma (MB) is an embryonic brain tumour that arises in the cerebellum. Using several MB cell lines, we have demonstrated that the chemotherapeutic drug etoposide induces a p53- and caspase-dependent cell death. We have observed an additional caspase-independent cell death mechanism involving delayed nuclear factor κB (NF-κB) activity. The delayed induction was controlled by a p53-dependent transcription step and the production of death receptors (especially CD95/Fas). We further demonstrated that in both MB and glioblastoma (GM) cell lines, in which the p53 pathway was not functional, no p65 activation could be detected upon etoposide treatment. MB cell lines that have mutations in p53 or NF-κB are either less sensitive (NF-κB mutant) or even completely resistant (p53 mutant) to chemotherapeutic intervention. The optimal cell death was only achieved when both p53 and NF-κB were switched on. Taken together, our results shed light on the mechanism of NF-κB activation by etoposide in brain tumours and show that the genetic background of MB and GM cells determines their sensitivity to chemotherapy and has to be taken into account for efficient therapeutic intervention.
Assuntos
Neoplasias Cerebelares/patologia , Etoposídeo/farmacologia , Meduloblastoma/patologia , NF-kappa B/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Cerebelares/enzimologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Meduloblastoma/enzimologia , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Receptores de Morte Celular/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
This study investigated possible integrated links in the neuroanatomical pathways through which the activity of neurones in the paraventricular nucleus and arcuate nucleus may modulate suppression of gonadotrophin-releasing hormone (GnRH) secretion during stressful situations. Double-label immunofluorescence and laser scanning confocal microscopy were used to examine the hypothalamic sections from the follicular phase ewes. Noradrenergic terminals were in close contact with 65.7 ± 6.1% corticotrophin-releasing hormone (CRH) and 84.6 ± 3.2% arginine vasopressin (AVP) cell bodies in the paraventricular nucleus but not with ß-endorphin cell bodies in the arcuate nucleus. Furthermore, γ-amino butyric acid (GABA) terminals were close to 80.9 ± 3.5% CRH but no AVP cell bodies in the paraventricular nucleus, as well as 60.8 ± 4.1%ß-endorphin cell bodies in the arcuate nucleus. Although CRH, AVP and ß-endorphin cell terminals were identified in the medial pre-optic area, no direct contacts with GnRH cell bodies were observed. Within the median eminence, abundant CRH but not AVP terminals were close to GnRH cell terminals in the external zone; whereas, ß-endorphin cells and terminals were in the internal zone. In conclusion, neuroanatomical evidence is provided for the ewe supporting the hypothesis that brainstem noradrenergic and hypothalamic GABA neurones are important in modulating the activity of CRH and AVP neurones in the paraventricular nucleus, as well as ß-endorphin neurones in the arcuate nucleus. These paraventricular and arcuate neurones may also involve interneurones to influence GnRH cell bodies in medial pre-optic area, whereas the median eminence may provide a major site for direct modulation of GnRH release by CRH terminals.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/anatomia & histologia , Hipotálamo/fisiologia , Ovinos/fisiologia , Estresse Fisiológico/fisiologia , Animais , Arginina Vasopressina , Hormônio Liberador da Corticotropina , Feminino , Receptores Adrenérgicos , Receptores de GABA , Estações do Ano , beta-EndorfinaRESUMO
Cardiovascular surgery with cardiopulmonary bypass (CPB) induces activation of blood coagulation and systemic inflammation involved in post-operative complications. Our study evaluated the impact of the minimal extracorporeal circulation (mini-CPB) system (Synergy, Sorin Group) on these functional aspects. Twenty patients were randomly assigned to standard CPB (n = 10) or to Synergy (n = 10). Platelet expression of PAC-1, and monocyte/granulocyte-platelet conjugates were evaluated by flow cytometry. A leukocyte-platelet adhesion index was calculated after cell number normalization. ELISAs were performed to measure IL-6 and TNF-alpha, thrombin-antithrombin III complexes (TAT), prothrombin fragments (F1+2), beta-thromboglobulin (beta-TG) and sP-selectin (sCD62P). Blood samples were drawn at the time of anesthesia (T1), at the end of CPB (T2), and at 4 (T3) and 24 hours (T4) after weaning from CPB. All patients were similar for clinical characteristics. When compared to standard CPB, the Synergy showed lower levels of the monocyte-platelet adhesion index at T2 (0.023 +/- 0.005 vs 0.063 +/- 0.013, P = 0.0092) and T4 (0.031 +/- 0.003 vs 0.055 +/- 0.005, P = 0.0017), TAT complexes at T2 (27.175 +/- 5.967 vs 86.592 +/- 5.415, P = 0.0005) and T3 (26.977 +/- 2.468 vs 45.146 +/- 4.365, P = 0.0041), F1+2 fragments at T2 (2.222 +/- 0.226 vs 4.249 +/- 0.292, P = 0.0009), and sP-selectin at T3 (115.17 +/- 19.623 vs 169.554 +/- 19.709, P = 0.0703) and T4 (108.542 +/- 6.429 vs 140.799 +/- 14.771, P = 0.0833). In summary, the Synergy exhibited a lower post-operative activation of blood coagulation, together with a reduced interaction between circulating monocytes and platelets.
Assuntos
Ponte de Artéria Coronária , Circulação Extracorpórea , Idoso , Coagulação Sanguínea , Plaquetas/fisiologia , Ponte de Artéria Coronária/instrumentação , Ponte de Artéria Coronária/métodos , Circulação Extracorpórea/instrumentação , Circulação Extracorpórea/métodos , Humanos , Pessoa de Meia-Idade , Monócitos/fisiologia , Selectina-P/sangueRESUMO
The transcription factor NF-kappaB (nuclear factor kappaB) regulates critical cellular processes including the inflammatory response, apoptosis and the cell cycle. Over the past 20 years many of the components of the NF-kappaB signalling pathway have been elucidated along with their functions. Recent research in this field has focused on the dynamic regulation and network control of this system. With key roles in so many important cellular processes, it is critical that NF-kappaB signalling is tightly regulated. Recently, single-cell imaging and mathematical modelling have identified that the timing of cellular responses may play an important role in the regulation of this pathway. p65/RelA (RelA) has been shown to translocate between the nucleus and cytoplasm with varying oscillatory patterns in different cell lines leading to differences in transcriptional outputs from NF-kappaB-regulated genes. Variations in the timing or persistence of these movements may control the maintenance and differential expression of NF-kappaB-regulated genes.
Assuntos
Proteínas I-kappa B/fisiologia , NF-kappa B/fisiologia , Células HeLa , Humanos , Cinética , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Oscilometria , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
The complement regulatory proteins CD55 and CD59 are expressed on the plasma membrane of human spermatozoa, whereas CD46 is only on the inner acrosomal membrane (IAM) which becomes surfaced exposed after the acrosome reaction when sperm assume fertilisation-competence. CD55 & CD59, two glycosylphosphatidylinositol (GPI)-anchored proteins, have been detected previously in some studies also in the acrosomal region of chemically fixed spermatozoa but never demonstrated at this site on unfixed spermatozoa. Dual labelling immunofluorescence and confocal microscopy on fresh unfixed spermatozoa, with minimal subsequent time to fixation, has shown CD55 to be markedly expressed on the IAM, more than on the plasma membrane. However, unlike for CD46, CD55 displayed patchy staining over the acrosome, with some variation between individual spermatozoa. All IAM-associated CD55 was localised within GM1-containing lipid rafts. CD59 was expressed also on the IAM, but in a pronounced granular pattern with more variation observed from one spermatozoa to another. Both CD55 & CD59 were released from the IAM by PI-PLC, demonstrating them to be GPI-anchored. Analysis of acrosome-reacted spermatozoal CD55 by Western blotting revealed a novel single 55 kDa protein lacking significant oligosaccharides susceptible to glycosidases. Antibody-induced membrane rafting and release of CD55 & CD59 in vitro may have influenced previous results. Significant coexpression of CD55 & CD46 on the IAM suggests some functional cooperation at this site.
Assuntos
Acrossomo/imunologia , Antígenos CD/metabolismo , Proteínas Inativadoras do Complemento/metabolismo , Western Blotting , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Membrana Celular/imunologia , Humanos , Masculino , Proteína Cofatora de Membrana/metabolismo , Microscopia de Fluorescência , Fosfatidilinositol Diacilglicerol-Liase/imunologia , Fosfoinositídeo Fosfolipase C , Espermatozoides/imunologia , Fatores de Tempo , Preservação de TecidoRESUMO
Signaling by the transcription factor nuclear factor kappa B (NF-kappaB) involves its release from inhibitor kappa B (IkappaB) in the cytosol, followed by translocation into the nucleus. NF-kappaB regulation of IkappaBalpha transcription represents a delayed negative feedback loop that drives oscillations in NF-kappaB translocation. Single-cell time-lapse imaging and computational modeling of NF-kappaB (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased IkappaBalpha transcription. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF-kappaB signaling may thus depend on number, period, and amplitude of oscillations.
Assuntos
Regulação da Expressão Gênica , NF-kappa B/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Simulação por Computador , Citoplasma/metabolismo , Etoposídeo/farmacologia , Retroalimentação Fisiológica , Células HeLa , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Modelos Biológicos , Inibidor de NF-kappaB alfa , Fosforilação , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição RelA , Transcrição Gênica , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Real-time imaging of the GH gene promoter linked to luciferase in living pituitary cells has revealed surprising heterogeneity and variety of dynamic patterns of gene expression. Cells treated with either forskolin or thyroid hormone generated a consistent and characteristic temporal response from cell populations, but detailed analysis of individual cells revealed different patterns. Approximately 25-26% of cells displayed no response, 25-33% of cells exhibited a sustained progressive rise in luciferase activity, and 41-50% showed a transient phasic, or oscillatory response, after given stimuli. In cells treated consecutively with the two stimuli, the population response to the second stimulus was augmented. Single-cell analysis revealed that this was partly due to an increased number of cells responding, but also that the prevalence of response patterns changed: cells that responded to an initial stimulus were more likely to respond subsequently in a progressive sustained manner. In conclusion, these studies have indicated that GH promoter activity in individual living pituitary cells is unstable and possibly stochastic, with dynamic variations from hour to hour. The prevalence of different temporal patterns of response to hormonal stimulation among a population of cells is altered by the endocrine history of those cells.
Assuntos
Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/metabolismo , Hipófise/citologia , Hipófise/fisiologia , Transcrição Gênica , Animais , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/farmacologia , Hormônio do Crescimento Humano/efeitos dos fármacos , Humanos , Luciferases/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Hipófise/efeitos dos fármacos , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Tri-Iodotironina/farmacologiaRESUMO
Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.
Assuntos
Cistatinas/metabolismo , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Western Blotting , Células Cultivadas , Cistatina C , Cistatinas/química , Cistatinas/imunologia , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Peso Molecular , Precursores de Proteínas/química , Precursores de Proteínas/imunologia , Transporte Proteico , Fatores de Tempo , TransfecçãoRESUMO
Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.
Assuntos
DNA Antissenso/genética , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/genética , Proteínas de Bactérias , Sítios de Ligação , Ligação Competitiva , Permeabilidade da Membrana Celular/efeitos dos fármacos , DNA Antissenso/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Sensibilidade e Especificidade , Estreptolisinas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células U937RESUMO
Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3'-P5' phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5'-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity.
Assuntos
Luciferases/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Transcrição Gênica/efeitos dos fármacos , Regiões 5' não Traduzidas/genética , Amidas , Animais , Besouros , Citomegalovirus/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Vetores Genéticos , Células HeLa , Humanos , Ácidos Fosfóricos , RNA Mensageiro/genética , Simplexvirus/genética , Tionucleotídeos , TransfecçãoRESUMO
Mcl-1 is a member of the Bcl-2 protein family, which has been shown to delay apoptosis in transfection and/or overexpression experiments. As yet no gene knockout mice have been engineered, and so there is little evidence to show that loss of Mcl-1 expression is sufficient to trigger apoptosis. U937 cells constitutively express the antiapoptotic protein Bcl-2; but during differentiation, in response to the phorbol ester PMA (phorbol 12 beta-myristate 13 alpha-acetate), Mcl-1 is transiently induced. The purpose of this investigation was to determine the functional role played by Mcl-1 in this differentiation program. Mcl-1 expression was specifically disrupted by chimeric methylphosphonate/phosphodiester antisense oligodeoxynucleotides to just 5% of control levels. The depletion of Mcl-1 messenger RNA (mRNA) and protein was both rapid and specific, as indicated by the use of control oligodeoxynucleotides and analysis of the expression of other BCL2 family members and PMA-induced tumor necrosis factor-alpha (TNF-alpha). Specific depletion of Mcl-1 mRNA and protein, in the absence of changes in cellular levels of Bcl-2, results in a rapid entry into apoptosis. Levels of the proapoptotic protein Bax remained unchanged during differentiation, while Bak expression doubled within 24 hours. Apoptosis was detected within 4 hours of Mcl-1 antisense treatment by a variety of parameters including a novel live cell imaging technique allowing correlation of antisense treatment and apoptosis in individual cells. The induction of Mcl-1 is required to prevent apoptosis during differentiation of U937 cells, and the constitutive expression of Bcl-2 is unable to compensate for the loss of Mcl-1. (Blood. 2000;96:1756-1763)
Assuntos
Apoptose/genética , Diferenciação Celular/efeitos dos fármacos , DNA Antissenso/farmacologia , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2 , Sequência de Bases , Western Blotting , Diferenciação Celular/genética , DNA Antissenso/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Microscopia Confocal , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Compostos Organofosforados/química , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Células U937RESUMO
A chimeric methylphosphonodiester/phosphodiester 15mer oligodeoxynucleotide of randomly selected sequence was observed to rapidly induce apoptosis in MOLT-4 and Jurkat E6 T lymphocytic leukaemia cells following intracytoplasmic delivery. A series of further methylphosphonate substitutions and mutations and truncations of the oligodeoxynucleotide served to establish that the phosphodiester-linked sequence CGGTA present in the 15mer was responsible for this biological activity. End-protected CpG oligodeoxy-nucleotide 5mers of sequence type CGNNN exhibited a range of apoptosis-inducing potencies, with CGTTA being the most active. The latter was shown to significantly reduce the rate of RNA synthesis in MOLT-4 cells within 1 h; DNA laddering and redistribution of phosphatidylserine to the outer surface of the plasma membrane were marked by 160 min and mitochondrial transmembrane potential collapsed over roughly the same time scale. Pro-caspase 8 was reduced within 130 min and the proteolytically activated caspase 8 substrate Bid was also down by this time, implicating release of cytochrome c from mitochondria by the active 15 kDa fragment of Bid. Substantial proteolytic activation of pro-caspase 3 was relatively delayed. These findings support a mitochondrial amplification mechanism for apoptosis triggered by CpG 5mers.
Assuntos
Apoptose/genética , Ilhas de CpG , Oligodesoxirribonucleotídeos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/metabolismo , Caspase 8 , Caspase 9 , Caspases/metabolismo , Membrana Celular/metabolismo , Precursores Enzimáticos/metabolismo , Humanos , Células Jurkat , Leucemia Linfoide , Potenciais da Membrana , Mitocôndrias/metabolismo , Fosfatidilserinas , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Uridina/metabolismoAssuntos
Oligodesoxirribonucleotídeos Antissenso/síntese química , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Transcrição Gênica/efeitos dos fármacos , Sequência de Bases , Quimera , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Eletroporação/métodos , Escherichia coli/enzimologia , Citometria de Fluxo/métodos , Humanos , Indicadores e Reagentes , Leucemia , Ácidos Nucleicos Heteroduplexes , Oligodesoxirribonucleotídeos Antissenso/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Ribonuclease H/metabolismo , Células Tumorais CultivadasRESUMO
A region of c-myc mRNA was identified which permitted very efficient antisense effects to be achieved in living cells using chimeric methylphosphonate--phosphodiester antisense effectors. Novel inosine--containing ribozymes (which cleave after NCH triplets) were directed to an ACA triplet within this region and delivered into living cells. No ribozyme intracellular activity could be identified. Very low ribozyme function was also observed in in vitro assays using a 1700nt substrate RNA.
Assuntos
DNA Antissenso/genética , Genes myc/genética , RNA Catalítico/metabolismo , Proteínas de Bactérias , Computadores , Escherichia coli/enzimologia , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , Ribonuclease H/metabolismo , Estreptolisinas/farmacologia , Células Tumorais CultivadasRESUMO
Antisense oligodeoxyribonucleotides (ODN) targeted against the breakpoint in BCR-ABL mRNA will specifically decrease BCR-ABL mRNA, provided cells are first permeabilised with streptolysin-O (SL-O). We used 18-mer chimeric methylphosphonodiester: phosphodiester linked (4-9-4) ODN complementary to 9 bases either side of the BCR-ABL junction to purge harvests ex vivo in three CML patients who remained completely Ph positive after multiple chemotherapy courses. After CD34+ cell selection and SL-O permeabilisation, harvests were purged with 20 microM ODN. After purging, all individual CFU-GM colonies grown from the two b3a2 breakpoint cases remained positive for BCR-ABL mRNA. In contrast, all 24 colonies grown from the b2a2 breakpoint case were BCR-ABL mRNA negative. Patients were conditioned with busulphan 16 mg/kg. The initial post-transplant course was uneventful, although the time to return to 0.5 x 10(9)/l neutrophils was slow at 25-51 days. Both chronic phase patients remain in haematological remission at +724 and +610 days, although each has cytogenetic evidence of relapse. The b2a2 accelerated phase patient died of myeloid blast transformation at day +91. The present SL-O-facilitated ODN purging strategy appears to be without significant toxicity, and offers considerable improvements in ODN delivery to the cytosol.
Assuntos
Proteínas de Fusão bcr-abl/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Oligonucleotídeos Antissenso/uso terapêutico , Condicionamento Pré-Transplante/métodos , Antígenos CD34 , Mobilização de Células-Tronco Hematopoéticas , Humanos , Leucaférese , Oligonucleotídeos Antissenso/administração & dosagem , Avaliação de Resultados em Cuidados de Saúde , Análise de Sequência de DNARESUMO
A 28-mer morpholino oligonucleotide analog was designed to hybridize to 8 bases of intron 1 and extend 2 bases beyond the translation initiation codon in exon 2 of the unspliced c-myc RNA transcript. Delivery of this compound into human chronic myeloid leukemia KYO1 cells, by streptolysin O permeabilization, resulted in almost total ablation of the 65 kDa c-MYC protein expression for at least 24 hours after treatment. An unexpected band with SDS-PAGE electrophoretic mobility indicating a protein of about 47 kDa was apparent on the 24-hour western blots that were developed using antibodies that recognize MYC protein C terminal epitopes. No inhibition of the approximately 2400 nt c-myc mRNA expression was observed by northern hybridization, a result of the inability of morpholino analogs to direct the activity of ribonuclease H. In fact, high molecular weight c-myc RNA species were found to have accumulated in antisense-treated KYO1 cells. Control sense and scrambled antisense morpholino analogs did not inhibit MYC protein expression or induce the appearance of the anomalous RNA and protein bands. Molecular analyses by RT-PCR and sequencing revealed that the morpholino antisense effector had (1) inhibited splicing of the c-myc pre-mRNA, (2) induced missplicing of the pre-mRNA, and (3) inhibited translation of normal spliced c-myc mRNA. Identical results were obtained with acute promyelocytic leukemia, acute lymphoblastic leukemia, and histiocytic lymphoma cell lines.