Increasing expression of the normal human CFTR cDNA in cystic fibrosis epithelial cells results in a progressive increase in the level of CFTR protein expression, but a limit on the level of cAMP-stimulated chloride secretion.
Cystic fibrosis (CF) results from mutations of the CF transmembrane conductance regulator (CFTR) gene and the consequent defective regulation of cAMP-stimulated Cl- permeability across epithelial cell apical membranes. Given that in vitro transfer of normal CFTR cDNA corrects this defect and that recombinant adenovirus (Ad) vectors can transfer the normal human CFTR cDNA in vivo, Ad vectors have significant potential in the development of effective strategies for CF gene therapy. One concern is whether CFTR overexpression achievable with Ad vectors may have untoward effects on cAMP-stimulated Cl- efflux. To address this, the CF pancreatic epithelial cell line CFPAC-1 was infected with increasing doses of AdCFTR, a recombinant Ad containing the normal CFTR cDNA, and analyzed for CFTR mRNA and protein levels and CFTR function. As the AdCFTR dose increased [multiplicity of infection (moi) 0-1,000], CFTR mRNA and protein levels increased. However, while CFTR function measured by cAMP-stimulated 36Cl- efflux was observed with low doses of the vector (moi 20), there was no further increase in CFTR function with increasing doses of AdCFTR (moi from 20 to 1,000). These data suggest that after AdCFTR-mediated gene transfer, epithelial cells limit the level of cAMP-stimulated Cl- secretion despite increasing levels of CFTR protein.
Rosenfeld
M A
MA
Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Rosenfeld
S J
SJ
Danel
C
C
Banks
T C
TC
Crystal
R G
RG
D000230:Adenocarcinoma / Q000150:complications / Q000473:pathology; D000260:Adenoviruses, Human; D000818:Animals; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000473:pathology; D000242:Cyclic AMP / Q000502:physiology*; D003550:Cystic Fibrosis / Q000150:complications / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D018076:DNA, Complementary / Q000235:genetics*; D004848:Epithelium / Q000378:metabolism / Q000473:pathology; D005822:Genetic Vectors; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000473:pathology; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D010190:Pancreatic Neoplasms / Q000150:complications / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012636:Secretory Rate; D014407:Tumor Cells, Cultured
C505032:CFTR protein, human; D002712:Chlorides; D018076:DNA, Complementary; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000242:Cyclic AMP