RESUMO
Cystic fibrosis (CF) patients bearing the premature translation termination mutation (nonsense mutation) W1282X present severe pulmonary and pancreatic disease, whereas patients carrying other nonsense mutations such as G542X, R553X, S1255X, R1162X, and W1316X show a severe pancreatic but mild pulmonary illness. CF gene expression was found absent in respiratory tissues with mutations R553X and W1316X, which led to the hypothesis that the absence of the gene product in the lung is more favorable than the presence of an altered one. We asked whether or not all the nonsense mutations characterized by mild pulmonary disease phenotypes do present the absence of CF gene expression. We therefore investigated gene expression at the mRNA level in respiratory cells obtained from nasal polyps from a patient homozygous for the R1162X mutation. Gene expression was studied by amplification with polymerase chain reaction of segments of the CF transmembrane conductance regulator cDNA that was obtained by reverse transcription of RNA. Semiquantitative analysis was performed by Northern analysis. By comparing the data obtained from polyps deriving from non-CF subjects and a CF patient homozygous for dF508 mutation, it is shown that no reduction of CF gene expression is evident in R1162X respiratory tissue. We conclude that CF nonsense mutations have heterogeneous mechanisms of gene expression.
Assuntos
Fibrose Cística/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Mutação , Pólipos Nasais/metabolismo , Mutação Puntual , RNA Mensageiro/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Neoplasias do Colo , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , Primers do DNA , DNA Complementar/metabolismo , Epitélio/metabolismo , Feminino , Expressão Gênica , Humanos , Leucócitos/metabolismo , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Deleção de Sequência , Células Tumorais CultivadasRESUMO
Cystic fibrosis is caused by defects in a chloride-transporting protein termed cystic fibrosis transmembrane conductance regulator (CFTR). This study presents an innovative procedure to evaluate expression of functional CFTR. The technique uses the potential-sensitive probe bis-(1,3-diethylthiobarbituric acid) trimethine oxonol or DiSBAC2(3), by single-cell fluorescence imaging. The DiSBAC2(3) method was first validated on the mouse mammary tumor cell line C127, stably expressing wild-type CFTR. Activation of protein kinase A by the cAMP-permeable analogue 8-Br-cAMP induced cell membrane depolarization consistent with expression of wild-type CFTR. The DiSBAC2(3) method is quick, simple, and reproducible, and does not require invasive cell loading procedures. The system was then applied to the cell model of the human lung tumor cell line A549, in which exogenous CFTR was expressed by infecting with the replication-deficient recombinant adenovirus AdCFTR. DiSBAC2(3) was able to detect the fraction of cells in which the expression of CFTR protein was confirmed by immunocytochemistry. The DiSBAC2(3) probe was also used in human nasal respiratory cells cultured in vitro, in which it efficiently discriminated between endogenous CFTR in normal and CF cells. Functional evaluation of CFTR function by the described method can be a useful tool to detect the expression of the CF gene transferred by adenoviral vectors for use in gene therapy trials.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/análise , Microscopia de Fluorescência/métodos , Adenoviridae/genética , Animais , Proteínas Quinases Dependentes de AMP Cíclico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ativação Enzimática , Corantes Fluorescentes , Técnicas de Transferência de Genes , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Neoplasias Pulmonares/química , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Mamárias Animais/química , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Potenciais da Membrana , Camundongos , Mutação , Pólipos Nasais/química , Pólipos Nasais/metabolismo , Pólipos Nasais/patologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The regulation of chloride conductance was investigated in the T84 human colon carcinoma cell line by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium. The permeable cAMP analog 8-Br-cAMP (100 microM) and the calcium ionophore ionomycin (1 microM) activate a chloride conductance. A prolonged (4 h) preincubation of cells with phorbol 12-myristate 13-acetate (100 nM) or with the diacylglycerol analog 1-oleoyl-2-acetyl-glycerol (100 microM): (i) down-modulates to almost zero the protein kinase C activity in the membranes; (ii) inhibits the activation of the chloride conductance mediated by 8-Br-cAMP but not by calcium; (iii) reduces the mRNA without changing the expression of the protein product of the cystic fibrosis gene. The data suggest that PKC is essential for the activation of the cAMP-dependent chloride conductance in T84 cells.
Assuntos
Cloretos/metabolismo , AMP Cíclico/farmacologia , Fibrose Cística/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Transporte Biológico , Canais de Cloreto , Neoplasias do Colo , Regulador de Condutância Transmembrana em Fibrose Cística , Diglicerídeos/farmacologia , Regulação para Baixo , Condutividade Elétrica , Fluorescência , Corantes Fluorescentes , Humanos , Ionomicina/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas Quinases/metabolismo , Compostos de Quinolínio , RNA Mensageiro/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
Gene transfer to the respiratory tract by replication-deficient adenoviruses is limited by the induction of inflammatory and immune responses. We previously demonstrated that a E1-E3-deleted recombinant adenovirus carrying the expression cassette for the cystic fibrosis gene (Ad.CFTR) upregulates the expression of the pro-inflammatory intercellular adhesion molecule-1 (ICAM-1) both in vitro and in vivo. In the present work we suggest a role for the nuclear factor-kB (NF-kB) in Ad.CFTR-dependent up-regulation of ICAM-1 in respiratory epithelial A549 cells. Specifically, Ad.CFTR induced translocation of NF-kB into the nucleus and binding to the proximal -228/-218 NF-kB consensus sequence on the ICAM-1 promoter. Ad.CFTR also stimulated a 13-fold increase in NF-kB-dependent expression of the CAT reporter gene under the control of a region of the ICAM-1 promoter, including the proximal NF-kB consensus sequence. The Ad.CFTR-dependent increase of ICAM-1 mRNA was abolished by inhibitors of NF-kB, such as N-acetyl-L-cysteine, pyrrolidine dithiocarbamate, parthenolide and the synthetic peptide SN50. All these inhibitors abolished both Ad.CFTR-induced NF-kB DNA binding and transactivating activities. These results indicate a critical role of NF-kB in the pro-inflammatory response elicited by replication-deficient adenoviral vectors in respiratory cells.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Terapia Genética/métodos , Molécula 1 de Adesão Intercelular/metabolismo , Pulmão/metabolismo , NF-kappa B/metabolismo , Acetilcisteína/farmacologia , Adenoviridae/genética , Antioxidantes/farmacologia , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/farmacologia , Humanos , NF-kappa B/antagonistas & inibidores , Peptídeos/farmacologia , Pirrolidinas/farmacologia , RNA Mensageiro/análise , Sesquiterpenos/farmacologia , Tiocarbamatos/farmacologia , Translocação Genética/efeitos dos fármacosRESUMO
OBJECTIVE: To assess the practicability and monitor the results of an active carrier testing program among relatives of cystic fibrosis (CF) patients. METHODS: Parents of CF patients in the Veneto and Trentino regions of northeastern Italy were asked to help recruit relatives aged between 18 and 45 years for CF mutation testing. RESULTS: Of 409 enrolled CF parents, 59.6% agreed to send to the CF Center family composition data of relatives up to the third degree, and 28.8% recruited relatives to carrier testing, providing names and addresses of those who, being contacted, expressed a willingness to be tested. The participation of parents was higher if they were young and had a child recently diagnosed with CF. Recruiting parents indicated 333 close relatives (59%) for testing. When contacted by the CF Center, 170 of these 333 (51%) attended for testing. The percentage of close relatives who spontaneously asked for the test was 5.4% before the carrier testing program started; it rose to 25.3% following the introduction of the active strategy. CONCLUSIONS: The participation of the parents of CF patients is an important factor affecting the utilization of testing by relatives. Besides this, the influence of a favorable medical and cultural context (participation of gynecologists and family doctors in testing programs, genetic education of the general population) has to be considered.
RESUMO
Administration of replication-deficient recombinant adenoviruses (Ad) designed as vectors for gene transfer to the airway tract of rats and monkeys has been associated with a dose-dependent inflammatory process a few days after viral exposure. Among the cellular mechanisms possibly involved, we investigated the expression of intercellular adhesion molecule-1 (ICAM-1), which is known to be induced by parainfluenza, adenovirus type 5 and respiratory syncytial viruses in vitro. To test this hypothesis, an Ad type 5-derived replication-deficient recombinant vector carrying the expression cassette for the cystic fibrosis gene (Ad.CFTR) was either incubated with A549 cells (a human-derived lung epithelial cell line) or instilled by bronchoscopic procedures into the airways of Rhesus monkeys. Ad.CFTR induced expression of ICAM-1 in A549 cells and up-regulated with time the basal levels of ICAM-1 mRNA in lung portions of Rhesus monkeys. These observations indicate that E1-E3-deleted replication-deficient adenoviral vectors are capable of inducing adhesion molecules known to play a role in inflammation.