Functional consequences of heterologous expression of the cystic fibrosis transmembrane conductance regulator in fibroblasts.
We studied the consequences of cystic fibrosis transmembrane conductance regulator (CFTR) expression in NIH-3T3 fibroblasts as a model for the effects of virally transduced CFTR expression in non-epithelial cells. Fibroblasts were infected with a retrovirus vector that contained the human CFTR and neor cDNAs. We selected and expanded G418-resistant clones that encompassed a range of CFTR expression. CFTR-mediated Cl-conductance function was measured as whole cell current, and CFTR protein was quantitated by immunoblot analysis. Overall, there was a good relationship between CFTR protein levels and CFTR-mediated Cl- conductance. Some clones had consistently high basal levels of CFTR-mediated Cl- conductance. This variation in function was partially explained by CFTR protein levels and was not due to clonal variation in cAMP metabolism. High levels of CFTR expression were associated with depolarization of fibroblast membrane potential. The CFTR-expressing clones with the largest basally active CFTR Cl- conductances and the most depolarized membrane potentials also exhibited slower growth rates. These results suggest that potential side effects of gene replacement therapy for cystic fibrosis include functional consequences of CFTR expression in non-epithelial cells.
Stutts
M J
MJ
Cystic Fibrosis Research Center, University of North Carolina, Chapel Hill 27599.
Gabriel
S E
SE
Olsen
J C
JC
Gatzy
J T
JT
O'Connell
T L
TL
Price
E M
EM
Boucher
R C
RC
D016475:3T3 Cells; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D002999:Clone Cells; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms; D000242:Cyclic AMP / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004553:Electric Conductivity / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
C505032:CFTR protein, human; D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000242:Cyclic AMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid