RESUMO
Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~45.9 kDa, ~37 kDa, and ~83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50>100 µg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2.1% after 24h. PA:LFn-GAL4:ASO transfection of non- or terminally-differentiated THP-1 cells and Vero cells resulted in 35.2 ± 19.1%, 36.4 ± 1.8% and 22.9 ± 6.9% (respectively) Synt5 expression after treatment with 200 pmol of ASO and demonstrated versatility. Nucleofection® with Stealth RNAi™ siRNA reduced HeLa Synt5 levels to 4.6 ± 6.1% whereas treatment with the PA:LFn-PKR:siRNA resulted in 8.5 ± 3.4% Synt5 expression after 24h (HeLa cells). These studies report for the first time an ASO and RNAi delivery system based upon protein toxin architecture that is devoid of polycations. This system may utilize regulated membrane back-fusion for the cytosolic delivery of ASOs and siRNA, which would account for the lack of toxicity observed. High delivery efficiency suggests further in vivo evaluation is warranted.
Assuntos
Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Técnicas de Silenciamento de Genes , Oligonucleotídeos Antissenso/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transfecção/métodos , Animais , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Oligonucleotídeos Antissenso/biossíntese , Proteínas Qa-SNARE/biossíntese , Proteínas Qa-SNARE/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Vero , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Splicing is an important and highly regulated step in gene expression. The ability to modulate it can offer a therapeutic option for many genetic disorders. Antisense-mediated splicing-correction approaches have recently been successfully exploited for some genetic diseases, and are currently demonstrating safety and efficacy in different clinical trials. Their application for the treatment of retinal dystrophies could potentially solve a vast panel of cases, as illustrated by the abundance of mutations that could be targeted and the versatility of the technique. In this review, we will give an insight of the different therapeutic strategies, focusing on the current status of their application for retinal dystrophies.
Assuntos
Regulação da Expressão Gênica , Terapia Genética/métodos , Oligonucleotídeos Antissenso/genética , Splicing de RNA/genética , RNA/genética , Distrofias Retinianas/terapia , Humanos , Oligonucleotídeos Antissenso/biossíntese , RNA/metabolismo , Distrofias Retinianas/genética , Distrofias Retinianas/metabolismoRESUMO
Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR), for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI) cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.
Assuntos
Amplificação de Genes , Oligonucleotídeos Antissenso/biossíntese , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Sondas de DNA , MicroRNAs/genética , Espectrometria de Massas por Ionização por Electrospray , Sequências de Repetição em TandemRESUMO
This study was purposed to investigate the inhibition of hTERT antisense oligodeoxynucleotide (ASODN) on the proliferation and telomerase activity in HL-60 cells and to explore the relativity between the telomerase activity and the expression of hTERT gene in HL-60 cells. After treated by hTERT ASODN the expression of hTERT was detected by RT-PCR, the morphological changes of HL-60 cells was observed with inverted microscopy, the cell proliferation was measured by MTT method, and the telomerase activity was determined with TRAP-ELISA and TRAP-PAGE. The results showed that after sealing hTERT gene with ASODN for 72 hours, the expression of hTERT gene was significantly inhibited, the cell growth was repressed and the ability of proliferation decreased, and the effect was specific in sequence and dependent in dose and time. OD(450-690) values were 2.648 +/- 0.42, 1.504 +/- 0.47, 1.223 +/- 0.39, 0.944 +/- 0.16 respectively, as the cells were treated with 0, 10, 20, 30 micromol/L ASODN for 72 hours. The difference was significant as compared 10, 20, 30 micromol/L groups with 0 micromol/L ASODN group respectively (P < 0.05), but the difference was no significant when compared 20 micromol/L SODN group (2.376 +/- 0.65) with untreated group (2.648 +/- 0.42) (P > 0.05). TRAP-PAGE detection revealed that comparing ASODN groups with SODN groups the telomerase image bands were decreased and least was found in groups of 30 +/- mol/L. It is concluded that the hTERT ASODN may inhibit the proliferation and down-regulate the telomerase activity in HL-60 cells by sealing the expression of hTERT gene.
Assuntos
Proliferação de Células/efeitos dos fármacos , Oligonucleotídeos Antissenso/genética , Telomerase/metabolismo , Células HL-60 , Humanos , Oligonucleotídeos Antissenso/biossíntese , Telomerase/biossíntese , Telomerase/genética , Telomerase/farmacologia , TransfecçãoRESUMO
The study was aimed to investigate the expression of Rac1 in human acute leukemic cell line HL-60 and effect of Rac1 on cell cycle progression and apoptosis. The mRNA expression of Rac1 in HL-60 cell line and normal human peripheral blood mononuclear cells (PBMNC) were examined by semi-quantitative RT-PCR. After transfection of HL-60 cells with different concentrations of Rac1 antisense oligodeoxynucleotide (ASODN) by means of FuGENE6, the survival, cell cycle, apoptosis of HL-60 cells were observed through MTT assay, FCM test, Wright-Giemsa, acridine orange/ethidium bromide (AO/EB) and Annexin V-FITC/PI staining test respectively. The results showed that Rac1 relative amount in HL-60 was 0.84 +/- 0.13, while it in the normal PBMNC was 0.26 +/- 0.1 (P < 0.01); the expression of Rac1 in HL-60 cells was significantly upregulated. Compared with sense oligodeoxynucleotide (SODN), HL-60 cell viability, after exposure to ASODN at a concentration of 2.0 g/L decreased, (73.7 +/- 5.0)% vs (93.2 +/- 3.0)% (P < 0.01), while the proportion of G(1) cells increased as (52.1 +/- 6.8)% vs (31.6 +/- 4.7)% (P < 0.05), the percentage of Annexin V positive cells increased, (19.2 +/- 2.1)% vs (4.1 +/- 1.7)% (P < 0.01), and HL-60 cells were observed to have formation of apoptotic bodies. The data presented indicate that Rac1 may be involved in regulation of HL-60 cell cycle and apoptosis, promote overproliferation of HL-60 cells and inhibit their apoptosis.
Assuntos
Apoptose/fisiologia , Ciclo Celular/fisiologia , Oligonucleotídeos Antissenso/biossíntese , Proteínas rac1 de Ligação ao GTP/biossíntese , Células HL-60 , Humanos , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
We describe a novel mechanism of viral RNA eradication by an oligodeoxynucleotide A (ODN A) directly in HIV virions. The ODN A consists of an antisense and a passenger strand, and was designed to target the polyp-urine tract (PPT) of HIV-1, a conserved region of the viral genome. It leads to HIV reverse transcriptase/ribonuclease H (RT/RNase H)-dependent degradation of the RNA in viral particles. Illimaquinone, a specific inhibitor of RNase H, activity of HIV RT/RNase H, prevents RNA cleavage. The effect of the ODN A is sequence-specific and the passenger strand is important, since a lack or alteration of this strand reduces the antiviral activity of the ODN. ODN A has a stronger antiviral effect compared to a control ODN CO, targeted to a site outside of the PPT. The pretreatment with ODN A strongly reduced the infectivity of virions in cell culture in the absence of any DNA carriers or detergents.
Assuntos
Antivirais/farmacologia , HIV-1/genética , Oligonucleotídeos Antissenso/farmacologia , Estabilidade de RNA/genética , RNA Viral/metabolismo , Vírion/genética , Sequência de Bases , Células Cultivadas , Transcriptase Reversa do HIV/metabolismo , HIV-1/patogenicidade , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/biossíntese , Ribonuclease H/metabolismo , Vírion/patogenicidadeRESUMO
OBJECTIVES: To study the inhibitory effect of specially synthesized oligodeoxynucleotide (SODN) on malignant phenotype of human hepatocellular carcinoma Hep3B cells by complementary binding to the fourth promoter of IGF-2 gene. METHODS: A SODN was synthesized according to the sequence of the fourth promoter of IGF-2 gene, and was then transfected into Hep3B cells which overexpressed IGF-2. IGF-2 gene transcription activity and protein expression were assayed by RT-PCR and Western blot methods. The effect on cell growth by SODN was estimated by MTT method, and the effect on cell cycle distribution was measured by flow cytometry. Colony formation assay was performed on 6-well tissue culture dishes. Alteration on mobility and invasiveness were studied used transwell plates. RESULTS: IGF-2 mRNA and protein levels in Hep3B cells transfected with SODN were significantly lower in comparison with those in control groups (Hep3B cells and Hep3B cells transfected with a control oligodeoxynucleotide). Results also showed that SODN did not have inhibitory effects on cell growth and mobility of Hep3B cells, but did have an effect on its colony formation and invasiveness. CONCLUSION: SODN has inhibitory effect on IGF-2 expression in Hep3B cells as a molecular switch, which partially alterates the malignant phenotype of this cell line.
Assuntos
Carcinoma Hepatocelular/patologia , Fator de Crescimento Insulin-Like II/biossíntese , Neoplasias Hepáticas/patologia , Oligonucleotídeos Antissenso/farmacologia , Humanos , Fator de Crescimento Insulin-Like II/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/farmacologia , Oligonucleotídeos Antissenso/biossíntese , Oligonucleotídeos Antissenso/genética , Fenótipo , Células Tumorais CultivadasRESUMO
OBJECTIVES: To construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase-2 (MMP2) and to study its inhibitory effects on the invasiveness and migratory capacity of hepatocellular carcinoma (HCC) cell line HepG2 in vitro. METHODS: Total RNA was extracted from HCC. Then a 500 bp fragment at the 5' end of the human MMP2 cDNA sequence was synthesized by polymerase chain reaction (PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV. With the resultant plasmid and the backbone plasmid pAdEasy-1, the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed. The adenovirus (Ad-MMP2AS) was packaged and amplified in the HEK 293 cells and the viral titer was checked by GFP. Using the Boyden chamber model, the influence of Ad-MMP2AS on the invasion ability of HepG2 cells was determined in vitro. RESULTS: The recombinant adenovirus vector carrying antisense MMP2 was constructed successfully and a strong green fluorescence was observed in HepG2 cells under a fluorescence microscope. The viral titer was 1 x 10(8); Ad-MMP2AS can effectively inhibit the penetrating capacity of HepG2 cells through Matrigel in vitro. CONCLUSION: The recombinant adenovirus with antisense MMP2 can effectively inhibit the invasiveness and migratory capacity of HepG2 in vitro and may have potential in treating HCC.
Assuntos
Adenoviridae/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Metaloproteinase 2 da Matriz/farmacologia , RNA Antissenso/farmacologia , Vetores Genéticos , Humanos , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Invasividade Neoplásica , Oligonucleotídeos Antissenso/biossíntese , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , RNA Antissenso/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Células Tumorais CultivadasRESUMO
A fundamental concept in computer science is that of the universal Turing machine, which is an abstract definition of a general purpose computer. A general purpose (universal) computer is defined as one which can compute anything that is computable. It has been shown that any computer which is able to simulate Boolean logic circuits of any complexity is such a general purpose computer. The field of DNA computing was founded in 1994 by Adleman's solution of a 7-bit instance of the Hamiltonian path problem. This work, as well as most of the subsequent experimental and theoretical investigations in the area, focused primarily upon the solution of NP-complete problems, which are a subset of the larger universal class of problems. In the present work a surface DNA computer capable of simulating Boolean logic circuits is demonstrated. This was done by constructing NOR and OR gates and combining them into a simple logic circuit. The NOR gate is one of the universal gates in Boolean logic, meaning that any other logic gate can be built from it alone. The circuit was solved using DNA-based operations, demonstrating the universal nature of this surface DNA computing model.
Assuntos
Computadores Moleculares , DNA/metabolismo , DNA/biossíntese , DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Ligases/metabolismo , Desnaturação de Ácido Nucleico , Oligonucleotídeos , Oligonucleotídeos Antissenso/biossíntese , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fosforilação , Reprodutibilidade dos TestesRESUMO
Thioredoxin (TRX) has a role in a variety of biological processes, including cytoprotection and the activation of transcription factors. Nerve growth factor (NGF) is a major survival factor of sympathetic neurons and promotes neurite outgrowth in rat pheochromocytoma PC12 cells. In this study, we showed that NGF induces TRX expression at protein and mRNA levels. NGF activated the TRX gene through a regulatory region positioned from -263 to -217 bp, containing the cAMP-responsive element (CRE). Insertion of a mutation in the CRE in this region abolished the response to NGF. NGF induced binding of CRE-binding protein to the CRE of the TRX promoter in an electrophoretic mobility shift assay. NGF also induced nuclear translocation of TRX. 2'-Amino-3'-methoxyflavone, an inhibitor of mitogen-activated protein kinase kinase, which is a known inhibitor of NGF-dependent differentiation in PC12 cells, suppressed the NGF-dependent expression and nuclear translocation of TRX. Overexpression of mutant TRX (32S/35S) or TRX antisense vector blocked the neurite outgrowth of PC12 cells by NGF. Overexpression of mutant TRX (C32S/C35S) suppressed the NGF-dependent activation of the CRE-mediated c-fos reporter gene. These results suggest that TRX plays a critical regulatory role in NGF-mediated signal transduction and outgrowth in PC12 cells.
Assuntos
Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tiorredoxinas/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neuritos/fisiologia , Oligonucleotídeos Antissenso/biossíntese , Oligonucleotídeos Antissenso/farmacologia , Oxirredução , Células PC12 , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , RNA Mensageiro/metabolismo , Ratos , Elementos de Resposta/efeitos dos fármacos , Elementos de Resposta/fisiologia , Transdução de Sinais/efeitos dos fármacos , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genéticaAssuntos
Antineoplásicos/uso terapêutico , Drogas em Investigação , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/terapia , Oligonucleotídeos Antissenso/biossíntese , Oligonucleotídeos Antissenso/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismoRESUMO
In human colorectal cancer, K-ras point mutations occur in approximately 40-50% of the cases, a frequency second only to pancreatic cancer (80-90%). Unlike pancreatic and lung cancers, however, the tumor-suppressive effect of antisense K-ras RNA expression has not been examined for colorectal cancers. A recombinant adenovirus vector expressing an antisense or sense K-ras gene fragment (AxCA-AS-K-ras or AxCA-S-K-ras) was first transduced into seven human colorectal cancer cell lines. Stable expression of antisense or sense K-ras RNA was detected by RNA blot analysis. Western blot analysis confirmed a reduction of up to 25% of K-ras-specific p21 protein in the antisense K-ras-transduced HCT-15 cells. In contrast to our previous findings on pancreatic cancer, the status of K-ras point mutations was not correlated with the growth-suppressive effect of the antisense K-ras vector: both the K-ras-mutation-positive and -negative colorectal cancer cell lines were suppressed for their growth in vitro. There was no growth-inhibitory effect on normal cells such as hepatocytes. Next, to test the efficacy in vivo, HCT-15 cells were inoculated subcutaneously into the left flank of SCID mice, and AxCA-AS-K-ras was injected intratumorally three times after the tumor mass was established. The infection of AxCA-AS-K-ras, but not the control AxCA-S-K-ras, significantly suppressed the growth of the HCT-15 subcutaneous tumor. This study shows that the adenovirus-mediated in vivo gene transfer of the antisense K-ras construct may be a useful therapeutic strategy for colorectal cancer.
Assuntos
Adenoviridae/genética , Neoplasias Colorretais/terapia , Genes ras/genética , Oligonucleotídeos Antissenso/genética , RNA/genética , Animais , Northern Blotting , Western Blotting , Linhagem Celular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Modelos Genéticos , Oligonucleotídeos Antissenso/biossíntese , Mutação Puntual , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Fatores de Tempo , Transdução Genética , Células Tumorais CultivadasRESUMO
Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Neurônios/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Tretinoína/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neoplásicas , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oligonucleotídeos Antissenso/biossíntese , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
This unit presents protocols that employ antisense oligodeoxynucleotides to reduce expression of target proteins in the brain. These oligonucleotides are generally designed to inhibit synthesis of a specific protein by hybridization to its mRNA. Because oligonucleotides show very poor penetration into the central nervous system (CNS) after systemic administration, they are either injected into the cerebrospinal fluid (CSF) or infused directly into the brain parenchyma. In this unit, the procedure most commonly used for delivering oligonucleotides continuously into CSF is outlined. In addition, a procedure is described for continuous infusion of oligonucleotides into a specific brain region, using the substantia nigra as an example.
Assuntos
Química Encefálica/genética , Regulação da Expressão Gênica/fisiologia , Marcação de Genes/métodos , Oligonucleotídeos Antissenso/biossíntese , Oligonucleotídeos Antissenso/genética , Animais , Injeções Intraventriculares , Masculino , Oligonucleotídeos Antissenso/administração & dosagem , Ratos , Ratos Sprague-DawleyRESUMO
Engagement of the B cell receptor of WEHI 231 immature B cells leads sequentially to a drop in c-Myc, to induction of the cyclin-dependent kinase inhibitor p27Kip1, and finally to apoptosis. Recently we demonstrated that the drop in c-Myc expression promotes cell death, whereas the induction of p27 has been shown to lead to growth arrest. In this paper, we demonstrate that increased p27 expression also promotes apoptosis of WEHI 231 B cells. The rescue of WEHI 231 cells by CD40 ligand engagement of its receptor prevented the increase in p27 induction. Inhibition of p27-ablated apoptosis induced upon expression of antisense c-myc RNA. Furthermore, specific induction of p27 gene expression resulted in apoptosis of WEHI 231 cells. Lastly, inhibition of expression of c-Myc, upon induction of an antisense c-myc RNA vector, was sufficient to induce increased p27 levels and apoptosis. Thus, these findings define a signaling pathway during B cell receptor engagement in which the drop in c-Myc levels leads to an increase in p27 levels that promotes apoptosis.
Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/imunologia , Imunoglobulina M/imunologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Supressoras de Tumor , Animais , Antígenos CD40/metabolismo , Ligante de CD40 , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/fisiologia , Inibidores Enzimáticos/farmacologia , Ligantes , Glicoproteínas de Membrana/fisiologia , Camundongos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/biossíntese , Oligonucleotídeos Antissenso/biossíntese , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , RNA Antissenso/biossíntese , RNA Antissenso/farmacologia , Transdução de Sinais/imunologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Transforming growth factor-beta1 (TGF-beta1) inhibits cell growth in susceptible cells by interacting with a family of protein kinases that control cell cycle progression. In the present study, we investigated the effects of TGF-beta1 on cyclin D1 expression and activity in COLO-357 human pancreatic cancer cells. TGF-beta1 increased cyclin D1 mRNA and protein levels. Nuclear runoff transcription and protein synthesis inhibition by cycloheximide revealed that this increase was, in part, due to increased cyclin D1 mRNA synthesis. Despite its stimulatory effects on cyclin D1 levels, TGF-beta1 inhibited cyclin D1-associated kinase activity and the growth of COLO-357 cells. Furthermore, suppression of cyclin D1 expression with a cyclin D1 antisense cDNA resulted in loss of TGF-beta1-mediated growth inhibition in association with reduced induction of cyclin D1, p21(C)(ip)(1) and plasminogen activator inhibitor-1 (PAI-1). Concomitantly, there was a marked decrease in the levels of the type I TGF-beta receptor (TbetaRI). Our findings suggest that in some cell types cyclin D1 expression may be important for TGF-beta1-mediated signaling and that cyclin D1 may be involved in the transcriptional regulation of TbetaRI.
Assuntos
Receptores de Ativinas Tipo I , Ciclina D1/antagonistas & inibidores , Ciclina D1/biossíntese , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Fator de Crescimento Transformador beta/farmacologia , Divisão Celular/efeitos dos fármacos , Ciclina D1/genética , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/biossíntese , Ciclinas/genética , Ciclinas/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Oligonucleotídeos Antissenso/biossíntese , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Neoplasias Pancreáticas/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Fator de Crescimento Transformador beta/fisiologia , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Previous studies with overexpressing wild-type or dominant negative nonvisual arrestins have established a role for these proteins in beta2-adrenergic receptor (beta2AR) internalization, desensitization, and resensitization. To validate and extend such findings, we employed an antisense strategy to target the nonvisual arrestins, arrestin-2 and arrestin-3, and determined the associated effects on the regulation of G protein-coupled receptor (GPCR) signaling. HEK293 cells stably expressing antisense constructs targeting arrestin-2 exhibited a selective reduction (approximately 50%) in arrestin-2 levels, while arrestin-3 antisense constructs resulted in reductions (>/=50%) in both arrestin-2 and arrestin-3 levels. Initial analysis of these cells demonstrated that a reduced level of arrestin expression resulted in a significant decrease in the extent of agonist-induced internalization of exogenously expressed beta2ARs, but had no effect on internalization of either m2 or m3 muscarinic acetylcholine receptors. Additional characterization involved assessing the role of arrestins in the regulation of endogenous GPCRs in these cells. Reduced arrestin levels significantly decreased the rate of endogenous beta2AR internalization, desensitization, and resensitization. Further analysis demonstrated that the desensitization of endogenous A2b adenosine and prostaglandin E2-stimulated receptors was also attenuated in cells with reduced arrestin levels. The effects on the beta2-adrenergic, A2b adenosine, and PGE2-stimulated receptors were similar among cell lines that exhibited either a selective reduction in arrestin-2 levels or a reduction in both arrestin-2 and -3 levels. These findings establish the utility of antisense approaches in the examination of arrestin-mediated GPCR regulation.
Assuntos
Arrestinas/genética , Proteínas de Ligação ao GTP/fisiologia , Oligonucleotídeos Antissenso/biossíntese , Fosfoproteínas/genética , RNA Mensageiro/biossíntese , Receptores de Superfície Celular/fisiologia , Adenilil Ciclases/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Animais , Arrestinas/antagonistas & inibidores , Arrestinas/biossíntese , Arrestinas/fisiologia , Células COS , Linhagem Celular Transformada , AMP Cíclico/biossíntese , Proteínas de Ligação ao GTP/genética , Humanos , Rim/citologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/biossíntese , Fosfoproteínas/fisiologia , Receptor Muscarínico M2 , Receptor Muscarínico M3 , Receptores Adrenérgicos beta 2/biossíntese , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Superfície Celular/genética , Receptores Muscarínicos/biossíntese , Receptores Muscarínicos/genética , TransfecçãoRESUMO
Retrons, bacterial retroelements found in Gram-negative bacteria, are integrated into the bacterial genome expressing a reverse transcriptase related to eukaryotic reverse transcriptase. The bacterial reverse transcriptases are responsible for the production of multicopy, single-stranded (ms) DNA consisting of a short single-stranded DNA that is attached to an internal guanosine residue of an RNA molecule by a 2',5'-phosphodiester linkage. Reverse transcriptases use an RNA transcript from the retrons, not only as primer, but also as template for msDNA synthesis. By studying the structural requirement, it was found that for msDNA synthesis an internal region of msDNA can be replaced with other sequences. msDNA can thus be used as a vector for in vivo production of an oligodeoxyribonucleotide of a specific sequence. Artificial msDNAs containing a sequence complementary to part of the mRNA for the major outer membrane lipoprotein of Escherichia coli effectively inhibited lipoprotein biosynthesis upon induction of msDNA synthesis. This is the first demonstration of in vivo synthesis of oligodeoxyribonucleotides having antisense function. Since we have previously demonstrated that bacterial retrons are functional in eukaryotes producing msDNA in yeast and in mouse NIH/3T3 fibroblasts, the present system may also be used to produce a specific oligodeoxyribonucleotide inside the cells to regulate eukaryotic gene expression artificially. We also describe a method to produce cDNA to a specific cellular mRNA using the retron system.
Assuntos
Proteínas de Transporte , DNA Antissenso/biossíntese , Proteínas de Escherichia coli , Lipoproteínas , Oligonucleotídeos Antissenso/biossíntese , Animais , Proteínas da Membrana Bacteriana Externa/genética , DNA de Cadeia Simples/biossíntese , Expressão Gênica , Camundongos , Dados de Sequência MolecularRESUMO
Recent advances in molecular biology especially in the area of rDNA, gene transfer, polymerase chain reaction and hybridoma techniques have provided the necessary molecular tools for the development of a new class of biopharmaceuticals. These biopharmaceuticals include antisense drugs, carbohydrate-based macromolecules and agents that interfere with apoptosis pathways. Cytokines and other immunomodulators also represent an exciting class of new therapeutic entities. The safety evaluation, efficacy, manufacturing and quality control of these complex biopharmaceuticals represent a challenge to the pharmacologist and toxicologist. Finally, the regulatory issues associated with the new biopharmaceuticals need to be addressed to insure the safety of these evolving therapeutic substances.
Assuntos
Biotecnologia/tendências , DNA Recombinante/genética , Apoptose/efeitos dos fármacos , Biotecnologia/legislação & jurisprudência , Carboidratos/biossíntese , Citocinas/farmacologia , Citocinas/uso terapêutico , Desenho de Fármacos , Técnicas de Transferência de Genes , Guias como Assunto , Hibridomas , Oligonucleotídeos Antissenso/biossíntese , Oligonucleotídeos Antissenso/normas , Reação em Cadeia da Polimerase , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , SegurançaRESUMO
Injection of messenger ribonucleic acid (mRNA) isolated from 1 alpha, 25-dihydroxyvitamin D3-treated osteoblast-like (PyMS) cells leads to an enhanced sodium-dependent phosphate (NadPi) transport in Xenopus laevis oocytes, when compared to untreated cells. After mRNA size fractionation, mRNA with an average size of 2.2-3.8 kilobases showed up to a 1.8-fold stimulation of NadPi transport encoding either directly a NadPi transporter(s) or proteins controlling their activity. No hybridization was observed in Northern blots with RNA from rat bone or PyMS cells with the recently cloned rat renal brush border NadPi transporter NaPi-2; hybrid depletion with a NaPi-2 antisense oligonucleotide did not abolish the PyMS mRNA-induced NadPi transport in oocytes. We present the first evidence for functional expression in Xenopus laevis oocytes of a new type of NadPi transport system in bone cells, which is different from the renal type.