No abstract

The DeltaF508 mutation reduces the amount of cystic fibrosis transmembrane conductance regulator (CFTR) expressed in the plasma membrane of epithelial cells. However, a reduced temperature, butyrate compounds, and "chemical chaperones" allow DeltaF508-CFTR to traffic to the plasma membrane and increase Cl(-) permeability in heterologous and nonpolarized cells. Because trafficking is affected by the polarized state of epithelial cells and is cell-type dependent, our goal was to determine whether these maneuvers induce DeltaF508-CFTR trafficking to the apical plasma membrane in polarized epithelial cells. To this end, we generated and characterized a line of polarized Madin-Darby canine kidney (MDCK) cells stably expressing DeltaF508-CFTR tagged with green fluorescent protein (GFP). A reduced temperature, glycerol, butyrate, or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)-stimulated transepithelial Cl(-) secretion across polarized monolayers. However, when the basolateral membrane was permeabilized, butyrate, but not the other experimental maneuvers, increased the CPT-cAMP-stimulated Cl(-) current across the apical plasma membrane. Thus butyrate increased the amount of functional DeltaF508-CFTR in the apical plasma membrane. Butyrate failed to stimulate transepithelial Cl(-) secretion because of inhibitory effects on Cl(-) uptake across the basolateral membrane. These observations suggest that studies on heterologous and nonpolarized cells should be interpreted cautiously. The GFP tag on DeltaF508-CFTR will allow investigation of DeltaF508-CFTR trafficking in living, polarized MDCK epithelial cells in real time.

Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, DeltaF508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, DeltaF508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 microM) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased DeltaF508-CFTR cell surface expression and DeltaF508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF.

No abstract

Cystic fibrosis is caused by mutations in the CFTR gene. The most common of these mutations, DF508, results in a protein that is not trafficked to the apical plasma membrane but instead is retained and degraded in the endoplasmic reticulum (ER) by the 26S proteosome. However, this protein is functional upon plasma membrane expression. It has been theoretically estimated that even a modest (∼10%) increase in CFTR-associated chloride conductance can be beneficial in a clinical setting. Thus, understanding basic CFTR biogenesis is important, and identification of prototypical compounds that can increase CFTR expression and trafficking is potentially useful in the development of novel therapeutic strategies to treat cystic fibrosis. We report that mitomycin C (MMC) elicits such a response by increasing CFTR mRNA and protein expression in T-84 and HT-29 cells at very low, non-cytotoxic, pharmacologically relevant concentrations (0.1 µM) leading to enhanced chloride secretion. Thus, MMC may be a useful compound for understanding CFTR regulation and biogenesis.