Increased functional cell surface expression of CFTR and DeltaF508-CFTR by the anthracycline doxorubicin.
ABSTRACT 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.
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ABSTRACT: The ATP-binding cassette (ABC) family of membrane transport proteins is the largest class of transporters in humans (48 members). The majority of ABC transporters function at the cell surface. Therefore, defective folding and trafficking of the protein to the cell surface can lead to serious health problems. The classic example is cystic fibrosis (CF). In most CF patients, there is a deletion of Phe508 in the CFTR protein (DeltaF508 CFTR) that results in defective folding and intracellular retention of the protein (processing mutant). A potential treatment for most patients with CF would be to use a ligand(s) of CFTR that acts a pharmacological chaperone to correct the folding defect. The feasibility of such an approach was first demonstrated with the multidrug transporter P-glycoprotein (P-gp), an ABC transporter, and a sister protein of CFTR. It was found that P-gps with mutations at sites equivalent to those found in CFTR processing mutants were rescued when they were expressed in the presence of drug substrates or modulators of P-gp. These compounds acted as pharmacological chaperones and functioned by promoting interactions among the various domains in the protein during the folding process. Several groups have attempted to identify compounds that could rescue the folding defect in DeltaF508 CFTR. The best compound identified through high-throughout screening is a quinazoline derivative (CFcor-325). Expression of DeltaF508 CFTR as well as other CFTR processing mutants in the presence of 1 muM CFcor-325 promoted folding and trafficking of the mutant proteins to the cell surface in an active conformation. Therefore, CFcor-325 and other quinazoline derivates could be important therapeutic compounds for the treatment of CF.Journal of Bioenergetics 01/2006; 37(6):501-7. · 1.60 Impact Factor
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ABSTRACT: Cystic fibrosis (CF) results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel localized at the plasma membrane of diverse epithelia. The most common mutation leading to CF, Delta F508, occurs in the first nucleotide-binding domain (NBD1) of CFTR. The Delta F508 mutation disrupts protein processing, leading to a decreased level of mutant channels at the plasma membrane and reduced transepithelial chloride permeability. Partial correction of the Delta F508 molecular defect in vitro is achieved by incubation of cells with several classes of chemical chaperones, indicating that further investigation of novel small molecules is warranted as a means for producing new therapies for CF. The yeast two-hybrid assay was used to study the effect of CF-causing mutations on the ability of NBD1 to self-associate and form dimers. A yeast strain demonstrating defective growth as a result of impaired NBD1 dimerization due to Delta F508 was used as a drug discovery bioassay for the identification of plant natural product compounds restoring mutant NBD1 interaction. Active compounds were purified and the chemical structures determined. The purified compounds were tested in epithelial cells expressing CFTR Delta F508 and the resulting effect on transepithelial chloride permeability was assessed using short-circuit chloride current measurements. Wild-type NBD1 of CFTR forms homodimers in a yeast two-hybrid assay. CF-causing mutations within NBD1 that result in defective processing of CFTR (Delta F508, Delta I507, and S549R) disrupted NBD1 interaction in yeast. In contrast, a CF-causing mutation that does not impair CFTR processing (G551D) had no effect on NBD1 dimerization. Using the yeast-based assay, we identified a novel limonoid compound (TS3) that corrected the Delta F508 NBD1 dimerization defect in yeast and also increased the chloride permeability of Fisher Rat Thyroid (FRT) cells stably expressing CFTR Delta F508. The establishment of a phenotype for the Delta F508 mutation in the yeast two-hybrid system yielded a simple assay for the identification of small molecules that interact with the mutant NBD1 and restore dimerization. The natural product compound identified using the system (TS3) was found to increase chloride conductance in epithelial cells to an extent comparable to genistein, a known CFTR activator. The yeast system will thus be useful for further identification of compounds with potential for CF drug therapy.Molecular Medicine 03/2002; 8(2):75-87. · 4.47 Impact Factor
- The Lancet 01/2002; 358(9298):2014. · 39.21 Impact Factor