Fhit regulates invasion of lung tumor cells.

In many types of cancers, the fragile histidine triad (Fhit) gene is frequently targeted by genomic alterations leading to a decrease or loss of gene and protein expression. Fhit has been described as a tumor suppressor gene because of its ability to induce apoptosis and to inhibit proliferation of tumor cells. Moreover, several studies have shown a correlation between the lack of Fhit expression and tumor aggressiveness, thus suggesting that Fhit could be involved in tumor progression. In this study, we explored the potential role of Fhit during tumor cell invasion. We first showed that a low Fhit expression is associated with in vivo and in vitro invasiveness of tumor cells. Then, we showed that Fhit overexpression in Fhit-negative highly invasive NCI-H1299 cells by transfection of Fhit cDNA and Fhit inhibition in Fhit-positive poorly invasive HBE4-E6/E7 cells by transfection of Fhit small interfering RNA induce, respectively, a decrease and an increase in migratory/invasive capacities. These changes in cell behavior were associated with a reorganization of tight and adherens junction molecules and a regulation of matrix metalloproteinase and vimentin expression. These results show that Fhit controls the invasive phenotype of lung tumor cells by regulating the expression of genes associated with epithelial-mesenchymal transition.

Quantitative videomicroscopic analysis of the sociologic behavior of non-invasive and invasive tumor cell lines.

To analyze the spatial distribution of tumor cell lines with different invasive properties, we used time-lapse videomicroscopic recordings associated with software programs we have developed for quantification. We observed that non-invasive tumor cells rapidly formed small clusters which aggregated to form larger clusters, whereas highly invasive tumor cells remained isolated and did not form clusters. An attraction index computed from a cellular automaton model was used to quantify the degree of attraction-repulsion between cells. The results suggest that the cluster formation by noninvasive cells is not related to a global attraction model and that the random (dispersed) distribution of invasive cells is not related to cell repulsion. According to these results, we can conclude that random cell movement combined with the intrinsic properties of cells explains the phenomenon of cluster formation.

Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells.

The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na(+) absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na(+), Mg(2+), P, S and Cl(-)) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR(inh)-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation.

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.

Quantitative cell dispersion analysis: new test to measure tumor cell aggressiveness.

Tumor progression requires the dispersion of epithelial cells from neoplastic clusters and cell invasion of adjacent stromal connective tissue. Aiming at demonstrating the precise relationships between cell dispersion and cell invasion, related respectively to expression of E-cadherin/catenin complex and matrix metalloproteinases (MMPs), we developed an original in vitro model of cell dispersion analysis. Our study reports the validation of this model that allowed us to analyze and quantify the cell cohesion level by means of time-lapse videomicroscopy and computer analysis based on the observation of spatial and temporal cell distribution. Our model was able to distinguish 2 groups among different human bronchial and mammary epithelial cells previously characterized for the expression of E-cadherin/catenin complex and MMPs and their invasive capacity in the Boyden chamber assay. The first group (16HBE14o(-), MCF-7, T47D) that expressed membranous E-cadherin and beta-catenin, and was negative for MMP-2 expression and non-invasive, displayed a highly cohesive pattern corresponding to a cluster spatial distribution. The second group (Beas2B, BZR, BZR-T33, MDA-MB-231, MDA-MB-435, BT549 and HS578T) that was invasive and showed lack of expression of E-cadherin and a cytoplasmic redistribution of beta-catenin, displayed a dispersed pattern corresponding to a random spatial distribution. Downregulation of E-cadherin by a blocking antibody induced a more random distribution. Conversely, expression of E-cadherin by cDNA transfection induced a cluster distribution. Moreover, tumor cell lines that co-expressed MT1-MMP and MMP-2 (Beas2B, BZR, BZR-T33, MDA-MB-435, BT549 and HS578T) showed a more dispersed pattern than tumor cell lines that did not express MMP-2 (MDA-MB-231). In conclusion, we demonstrated that the spatial group behavior of cell lines, i.e., their cohesion/dispersion ability, reflects their invasive properties. Thus, this model of cell dispersion analysis may represent a new test to measure tumor cell aggressiveness.

X-ray microanalysis of airway surface liquid collected in cystic fibrosis mice.

The airway surface liquid (ASL) that lines the airway surface epithelium plays a major role in airway antibacterial defense and mucociliary transport efficiency, two key factors in cystic fibrosis (CF) disease. A major difficulty is to collect ASL in native conditions without stimulation or alteration of the underlying airway epithelium. Using a cryoprobe specifically adapted to collect native ASL from the tracheal mouse surface, we analyzed by X-ray microanalysis the complete ASL and plasma ion content in Cftr(tm1Hgu)/Cftr(tm1Hgu) mice compared with that in control littermates. ASL ion content from eight Cftr(tm1Hgu)/Cftr(tm1Hgu) mice and eight control littermates did not appear significantly different. The mean (+/-SE) concentrations were 2,352 +/- 367 and 2,058 +/- 401 mmol/kg dry weight for Na, 1,659 +/- 272 and 1,448 +/- 281 mmol/kg dry weight for Cl, 357 +/- 57 and 337 +/- 38 mmol/kg dry weight for S, 1,066 +/- 220 and 787 +/- 182 mmol/kg dry weight for K, 400 +/- 82 and 301 +/- 58 mmol/kg dry weight for Ca, 105 +/- 31 and 105 +/- 20 mmol/kg dry weight for Mg, 33 +/- 15 and 29 +/- 9 mmol/kg dry weight for P in non-CF and CF mice, respectively. This cryotechnique appears to be a promising technique for analyzing the complete elemental composition of native ASL in CF and non-CF tissues.

Improved activity of an actin-resistant DNase I variant on the cystic fibrosis airway secretions.

In cystic fibrosis (CF), actin and DNA originating from inflammatory cells contribute to the thickness of airway secretions. Actin can bind to DNA-rich fibers and potently inhibit the enzymatic activity of rhDNase. The in vitro effects of the actin-resistant rhDNase variant (A114R) were analyzed and compared with those of the wild-type rhDNase. Frozen and thawed CF airway secretions were incubated for 30 min with different concentrations (0.1, 0.5, 1, 5, or 10 microg/ml) of either actin-resistant rhDNase or wild-type rhDNase. We observed that both the wild-type and the actin-resistant rhDNase significantly decreased (p < 0.05 and p < 0.001, respectively) the airway secretion viscosity. The decrease in airway secretion viscosity was significant even at low concentrations (0.1 microg/ml) of the actin-resistant variant. Incubation with the actin-resistant variant resulted in a significant decrease (p < 0.02) of the airway secretion contact angle and cough transport. A significantly higher (p < 0.01) increase in contact angle and cough transport of airway secretions was observed at 10 microg/ml with the actin-resistant variant as compared with the wild-type rhDNase. The present study had demonstrated that the actin-resistant rhDNase variant (A114R) has an enhanced capacity to improve the physical properties and cough transport of airway secretions from patients with cystic fibrosis.

Motogenic effect of recombinant HGF on airway epithelial cells during the in vitro wound repair of the respiratory epithelium.

Cell migration is the earliest mechanism involved in the wound repair process of the respiratory epithelium and could be potentially enhanced by growth factors. In the present work, we investigated the localisation of the hepatocyte growth factor (HGF) receptor (c-Met) during wound repair and evaluated the effect of recombinant HGF (rHGF) on cell migration by using an in vitro model of airway epithelial wound repair. By using immunohistochemical methods, we observed that the immunoreactivity of the c-Met proto-oncogene was increased in epithelial cells engaged in the process of tissue repair. The incubation of wounded cultures with increasing concentrations of rHGF (0.2, 2, 20, and 200 ng/ml) induced a significant (P < 0.02) dose-dependent effect on the wound repair index, with a maximum effect produced at 20 ng/ml (+31.3%). The cell migration speed reached 50.2 micrometer/h at this concentration, compared to 20.4 micrometer/h in the absence of rHGF. No significant effect on cell proliferation was observed in the repairing area in the presence of rHGF. These results suggest that rHGF is able to improve the wound repair process of the airway epithelium by increasing cell migration.

Beta(1)-integrins are involved in migration of human fetal tracheal epithelial cells and tubular morphogenesis.

Development of human fetal airways requires interaction of the respiratory epithelium and the extracellular matrix through integrins. Nevertheless, the specific roles of beta(1)-integrins during development and tubular morphogenesis are still unknown. To analyze beta(1)-integrin localization and influence during migration, we developed a model of human fetal tracheal explants growing on collagen and overlaid with a second layer of collagen to form a sandwich. In this configuration, cord and tubule formation proceeded normally but were inhibited by incubation with anti-beta(1)-integrin subunit antibodies. On a collagen matrix, beta(1)-integrins were immunolocalized on the entire plasma membrane of migrating epithelial cells and almost exclusively on the basal plasma membrane of nonmigratory epithelial cells. In a sandwich configuration, beta(1)-integrins became detectable in the cytoplasm of epithelial cells. Coating cultures with collagen transiently altered the morphology of migrating cells and their speed and direction of migration, whereas incubation with anti-beta(1)-integrin subunit antibodies irreversibly altered these parameters. These observations suggest that the matrix environment, by modulating beta(1)-integrin expression patterns, plays a key role during tubular morphogenesis of human fetal tracheal epithelium, principally by modulating epithelial cell migration.

Pseudomonas aeruginosa virulence factors delay airway epithelial wound repair by altering the actin cytoskeleton and inducing overactivation of epithelial matrix metalloproteinase-2.

To investigate the role of P. aeruginosa virulence factors in the repair of human airway epithelial cells (HAEC) in culture, we evaluated the effect of stationary-phase supernatants from the wild-type strain PAO1 on cell migration, actin cytoskeleton distribution, epithelial integrity during and after repair of induced wounds, and the balance between matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). PAO1 supernatant altered wound repair by slowing the migration velocity in association with altered actin cytoskeleton polymerization in the lamellipodia of migrating airway epithelial cells and delaying or inhibiting the restoration of epithelial integrity after wound closure. PAO1 virulence factors overactivated two of the gelatinolytic enzymes, MMP-2 and MMP-9, produced by HAEC during repair. During HAEC repair in the presence of PAO1 virulence factors, enhanced MMP-2 activation was associated with decreased rates of its specific inhibitor TIMP-2, whereas enhanced MMP-9 activation was independent of changes of its specific inhibitor TIMP-1. These inhibitory effects were specific to P. aeruginosa elastase-producing strains (PAO1 and lipopolysaccharide-deficient AK43 strain); supernatants from P. aeruginosa strain elastase-deficient PDO240 and Escherichia coli strain DH5alpha had no inhibitory effect. To mimic the effects of P. aeruginosa, we further analyzed HAEC wound closure in the presence of increasing concentrations of activated MMP-9 or MMP-2. Whereas increasing concentrations of active MMP-9 accelerated repair, excess activated MMP-2 generated a lower migration velocity. All these data demonstrate that P. aeruginosa virulence factors, especially elastase, may impede airway epithelial wound closure by altering cell motility and causing an imbalance between pro- and activated forms of MMP-2.

Vimentin contributes to human mammary epithelial cell migration.

Vimentin expression in human mammary epithelial MCF10A cells was examined as a function of their migratory status using an in vitro wound-healing model. Analysis of the trajectories of the cells and their migratory speeds by time lapse-video microscopy revealed that vimentin mRNA and protein expression were exclusively induced in cells at the wound's edge which were actively migrating towards the center of the lesion. Actin labeling showed the reorganization of actin filaments in cells at the wound's edge which confirmed the migratory phenotype of this cell subpopulation. Moreover, the vimentin protein disappeared when the cells became stationary after wound closure. Using cells transfected with the vimentin promoter controlling the green fluorescent protein gene, we also demonstrated the specific activation of the vimentin promoter in the migratory cells at the wound's edge. Transfection of the antisense vimentin cDNA into MCF10A cells clearly reduced both their ability to express vimentin and their migratory speed. Taken together, these observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.

Airway epithelial cell migration dynamics. MMP-9 role in cell-extracellular matrix remodeling.

Cell spreading and migration associated with the expression of the 92-kD gelatinase (matrix metalloproteinase 9 or MMP-9) are important mechanisms involved in the repair of the respiratory epithelium. We investigated the location of MMP-9 and its potential role in migrating human bronchial epithelial cells (HBEC). In vivo and in vitro, MMP-9 accumulated in migrating HBEC located at the leading edge of a wound and MMP-9 expression paralleled cell migration speed. MMP-9 accumulated through an actin-dependent pathway in the advancing lamellipodia of migrating cells and was subsequently found active in the extracellular matrix (ECM). Lamellipodia became anchored through primordial contacts established with type IV collagen. MMP-9 became amassed behind collagen IV where there were fewer cell-ECM contacts. Both collagen IV and MMP-9 were involved in cell migration because when cell-collagen IV interaction was blocked, cells spread slightly but did not migrate; and when MMP-9 activation was prevented, cells remained fixed on primordial contacts and did not advance at all. These observations suggest that MMP-9 controls the migration of repairing HBEC by remodeling the provisional ECM implicated in primordial contacts.

Pseudomonas aeruginosa internalization by human epithelial respiratory cells depends on cell differentiation, polarity, and junctional complex integrity.

Internalization of Pseudomonas aeruginosa by epithelial respiratory cell lines has been suggested to be dependent on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Because we have observed intracellular (IC) P. aeruginosa only in cells that do not express apical CFTR, we addressed the question of whether bacterial internalization by epithelial cells depends on the degree of cell differentiation and polarity. Internalization of piliated P. aeruginosa PAO-1 and PAK by human epithelial respiratory cells in primary culture and by the 16 human bronchial epithelial 14o- cell line cultured either on thick collagen gels or on thin collagen films was evaluated by the gentamicin exclusion assay. Cells cultured on thick gels were differentiated, polarized, and tight. They exhibited CFTR at their apical membranes, expressed beta1 integrins at their basal membranes, excluded lanthanum nitrate, and uniformly expressed ZO-1 protein. In contrast, in cells cultured on thin films, CFTR was present mainly in the cytoplasm, whereas beta1 integrins were detected at apical membranes. Most cells cultured on thin films did not exclude lanthanum nitrate and rarely expressed ZO-1 protein. Cells grown on thick and thin collagen substrates differed markedly in bacterial internalization: no IC bacteria could be detected in cells cultured on gels, whereas high IC bacterial concentrations were isolated from cells cultured on thin films. Treatment of cells cultured on thin films with ethylenediaminetetraacetic acid, to disrupt intercellular junctions further, significantly enhanced P. aeruginosa internalization. Our results suggest that P. aeruginosa internalization by epithelial respiratory cells does not depend on CFTR protein expression at the epithelial cell surface but rather on cell polarity and junctional complex integrity.

Ion composition and rheology of airway liquid from cystic fibrosis fetal tracheal xenografts.

The composition of airway surface liquid (ASL) is partly determined by active ion and water transport through the respiratory epithelium. It is usually stated that in cystic fibrosis (CF), CF transmembrane conductance regulator protein abnormality results in imbalanced ion composition and dehydration of ASL, leading to abnormal rheologic and transport properties. To explore the relationship between ion composition, water content, and viscosity of airway liquid (AL), we used a human xenograft model of fetal airways developed in severe combined immunodeficiency (SCID) mice. Six non-CF and six CF portions of fetal tracheas were engrafted subcutaneously in the flanks of SCID mice raised in pathogen-free conditions. AL accumulated in the closed cylindric grafts was harvested 9 to 17 wk after implantation. At the time of AL sampling, all tracheal grafts displayed well-differentiated pseudostratified surface epithelium and submucosal glands. The viscosity of AL was measured using a controlled-stress rheometer. The ion composition of AL was quantified by X-ray microanalysis. No significant difference was observed for AL viscosity between non-CF (0.6 +/- 0.5 Pa. s) and CF (0.2 +/- 0.1 Pa. s) samples. In AL from non-CF and CF samples, the ion concentrations were Na: 63.9 +/- 7.6, 79.7 +/- 11.6; Cl: 64.9 +/- 13.2, 82.6 +/- 15.7; Mg: 1.9 +/- 0.3, 2.2 +/- 0.4; S: 4.9 +/- 1. 3, 4.8 +/- 0.5; K: 2.4 +/- 0.5, 3.2 +/- 1.6; and Ca: 1.2 +/- 0.3, 2.6 +/- 0.8 mmol/liter, respectively. The ion composition of AL from CF versus non-CF xenografts was not significantly different. These results suggest that prior to inflammation and infection, the viscosity and ion composition of the fetal AL do not differ in CF and non-CF.

X-ray microanalysis of native airway surface liquid collected by cryotechnique.

The airway surface liquid (ASL) that lines the surface epithelium of the tracheobronchial tree is of vital importance to the airway defence against microbial invasion and damage due to environmental factors. Little is known about the ASL collected in situ in native conditions, owing to difficulties in collecting ASL without causing damage to the airway mucosa. We have developed a method to collect and analyse the elemental composition of tracheal ASL in pathogen-free mice. A specially designed cryoprobe, adapted to the internal curvature of the mouse trachea, was used to collect the native ASL from the tracheal surface. The complete ASL elemental composition including [Na] = 5.5 +/- 0.3, [Cl] = 1.3 +/- 0.3, [K] = 1.1 +/- 0.2, [Ca] = 1.2 +/- 0.3, [P] = 1.5 +/- 0.8, [S] = 1.7 +/- 0.4 and [Mg] = 1.3 +/- 0.4 mmol L-1 was determined by X-ray micro-analysis. We demonstrate here that the technique that we used for ASL collection maintained perfectly the airway epithelial integrity and functionality.

Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro.

Accumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the deltaF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1beta and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured deltaF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl- concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl- concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients.

Improvement of cystic fibrosis airway mucus transportability by recombinant human DNase is related to changes in phospholipid profile.

The aim of this study was to test whether changes in mucus surface properties by rhDNase treatment could be related to an increased recovery of phospholipids. Purulent sputa from 18 patients with cystic fibrosis (CF) were incubated with either rhDNase (4 microg/ml) or control excipient. The incubation of mucus samples with rhDNase induced a significant increase (p < 0.002) in the sol phase proportion (33.7 +/- 24.0%) compared with that obtained with excipient (12.6 +/- 12.4%). Phospholipids were recovered in significantly (p < 0.05) greater amounts from both mucus gel and sol phases after incubation with rhDNase. The phosphatidylglycerol content of mucus sol phase was significantly increased by rhDNase (p < 0.03), as well as the mucus gel phase surface properties and transport by ciliary activity and by cough (p < 0.05). The improvement of mucus gel surface properties and transport capacity by ciliary activity were significantly related to the increased recovery of phosphatidylglycerol (r = -0.74, p < 0.03 and r = 0.94, p < 0.05, respectively). These results suggest that rhDNase is able to increase the free water content and alter the phospholipid profile of mucus, with a related improvement in CF mucus transportability.

Analysis of image sequences in fluorescence videomicroscopy of stationary objects.

Fluorescence videomicroscopy allows the temporal behavior of biological specimens to be studied at the cellular level. We describe two types of methods that can be used for extracting quantitative information from image sequences: the modeling approach, which is mainly local, and multivariate statistical analysis, which provides a global approach. The potentials for use of these two methods are illustrated through a simulation example and actual examples dealing with the study of chloride secretion by airway epithelial cells. We define some guidelines for making a choice between these two approaches, bearing in mind that a blend of these two methodologies is also possible.

ATP depletion induces a loss of respiratory epithelium functional integrity and down-regulates CFTR (cystic fibrosis transmembrane conductance regulator) expression.

To mimic the effect of ischemia on the integrity of airway epithelium and expression of cystic fibrosis transmembrane conductance regulator (CFTR), we induced an ATP depletion of the respiratory epithelium from upper airway cells (nasal tissue) and human bronchial epithelial 16HBE14o- cell line. Histological analysis showed that 2 h of ATP depletion led to a loss of the epithelium integrity at the interface between basal cells and columnar cells. The expression of connexin 43 (Cx43, subunit of the gap junctions) and desmoplakins 1 and 2 (DPs 1 and 2, major components of the desmosomes) proteins was inhibited. After 90 min of ATP depletion, a significant decrease of the transepithelial resistance (25%) was observed but was reversible. Similar results were obtained with the 16HBE14o- human bronchial epithelial cell line. ATP depletion led to actin filaments depolymerization. The expression of the mature CFTR (170 kDa) and fodrin proteins at the apical domain of the ciliated cells was down-regulated. The steady-state levels of CFTR, Cx43, DPs 1 and 2 mRNAs, semiquantified by RT-polymerase chain reaction kinetics, remained constant throughout ATP depletion in nasal tissue as in the homogeneous cell population of 16HBE14o- human bronchial epithelial cell line. This suggests that the down-regulation of these proteins might be posttranscriptional. The intercellular diffusion through gap junctions of Lucifer dye was completely inhibited after 90 min of ATP depletion but was reversible. The volume-dependent and the cAMP-dependent chloride secretion were inhibited in a nonreversible way. Taken together, these results suggest that an ATP depletion in human airway epithelium, mimicking ischemia, may induce a marked alteration in the junctional complexes and cytoskeleton structure concomitantly with a loss of apical CFTR expression and chloride secretion function.

Induction of a cAMP-stimulated chloride secretion in regenerating poorly differentiated airway epithelial cells by adenovirus-mediated CFTR gene transfer.

In cystic fibrosis (CF), the airway epithelium is in the process of injury and regeneration. In the context of the CF gene therapy, we previously reported that regenerating poorly differentiated (PD) cells of human airway epithelium represent preferential cell targets for recombinant adenoviral gene vectors. To define whether PD non-CF and CF epithelial cells possess a functional cystic fibrosis transmembrane conductance regulator protein (CFTR) chloride channel, we analyzed the CFTR expression and the regulation of chloride secretion under cyclic (c)AMP stimulation in these regenerating PD epithelial cells of non-CF and CF airway tissue. Moreover, we studied the effects of CFTR gene transfer mediated by a replication-defective adenovirus containing the wild-type CFTR gene (AdCFTR) on CFTR expression and on cAMP-stimulated chloride secretion. Distribution of the CFTR protein was evaluated in regenerating PD airway cells by light fluorescence microscopy and scanning laser confocal microscopy. The cAMP-mediated regulation of cell membrane chloride secretion was investigated using the whole-cell patch clamp and SPQ (6-methoxy-N-[3-sulfopropyl]quinolinium) techniques. Compared with the absence of CFTR expression and cAMP-regulated chloride secretion in nontransduced regenerating PD cells of either non-CF or CF origin, transduction with AdCFTR induces a CFTR expression and a cAMP-regulated stimulation of the cell membrane chloride secretion in the regenerating PD cells. These results suggest that, out of the context of CF, remodeled and poorly differentiated airway epithelium may present abnormalities in ion transport. Moreover, our data suggest that, in the context of CF gene therapy, adenoviral vectors can be efficient in correcting, at least partially, the chloride secretion defect in the remodeled CF airway epithelium.