Epithelial cell specific properties and genetic complementation in a ΔF508 cystic fibrosis nasal polyp cell line

Abstract

Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to lose their tight junctions with multiple subcultures. To elucidate ion transport and protein trafficking in CF epithelial cells, a polar cell line with apical and basolateral compartments will facilitate analysis of the efficacy of different gene therapy strategies in a "tight epithelium" in vitro. This study investigates the genotypic and phenotypic properties of a CF nasal polyp epithelial, delta F508 homozygote, cell line that has tight junctions pre-crisis. The cells (sigma CFNPE14o-) were transformed with an origin-of-replication defective SV40 plasmid. They develop transepithelial resistance in Ussing chambers and are defective in cAMP-dependent Cl- transport as measured by efflux of radioactive Cl-, short circuit current (Isc), or whole-cell patch clamp. Stimulation of the cells by bradykinin, histamine, or ATP seems to activate both K(+)- and Ca(+2)-dependent Cl- transport. Measurement of 36Cl- efflux following stimulation with A23187 and ionomycin indicate a Ca(+2)-dependent Cl- transport. Volume regulatory capacity of the cells is indicated by cell swelling conductance. Expression of the CF transmembrane conductance regulator mRNA was indicated by RT-PCR amplification. When cells are grown at 26 degrees C for 48 h there is no indication of cAMP-dependent Cl- as has been previously indicated in heterologous expression systems. Antibodies specific for secretory cell antigens indicate the presence of antigens found in goblet, serous, and mucous cells; in goblet and serous cells; or in goblet and mucous cells; but not antigens found exclusively in mucous or serous cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cl Transport in Complemented CF Bronchial Epithelial Cells Correlates with CFTR mRNA Expression Levels

“…Antibodies to the SV40 large T antigen, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The cells were fixed and stained as described previously with a FITC-labeled secondary antibody [2,19,20,22,25]. Cells were visualized by fluorescence microscopy (Olympus IM-2) at 600× magnification.…”

“…A number of immortalized airway epithelial cell lines generated in the past have been critical for enhancing the understanding of the pathways responsible for CF pathology [2,19–30]. Transformed heterologous cells transfected with wt or mutant CFTR cDNA have also been widely used for biochemical studies [31–35].…”

“…Therefore, this study has also undertaken the task of generating a stable CF airway epithelial cell line complemented with the 6.2 kb wtCFTR cDNA construct. The parental CFBE41o− cell line is polarized and was used to derive recombinant subclones that were transfected with an episomal expression vector containing wt or ΔF508 CFTR [2,50]. Subclones were chosen based on the level of transgene-derived CFTR mRNA expression, i.e., the clones expressing the highest levels of CFTR mRNA.…”

Lack of correlation between CFTR expression, CFTR Cl? currents, amiloride-sensitive Na+ conductance, and cystic fibrosis phenotype

“…CFTR mRNA, CFTR Cl − Currents, and CF Phenotypeairway epithelial cells. 22,24 Expression of CFTR mRNA in the CF airway cell line transfected with an episomal high copy number plasmid (CFDE/CFTR) 23 was about 300 times higher than that of non-CF epithelial cells (16HBE14o-). However, functional expression of CFTR in the form of cAMP-activated Cl − conductance was comparable in both cell lines.…”

Reduction in the adherence of Pseudomonas aeruginosa to native cystic fibrosis epithelium with anti-asialoGM1 antibody and neuraminidase inhibition

“…CFNPE9o cells [16] were cultured for 48 h to 30% confluence in MEM containing 10% foetal calf serum, 1% penicillin/streptomycin, 1% L-glutamine, and 1% nonessential amino-acids. Monolayers were trypsinized for 5±10 min with 20 mL trypsin/ethylenediaminetetraacetic acid, following which 2 mL foetal calf serum was added, and cells were washed in PBS.…”