Novel human bronchial epithelial cell lines for cystic fibrosis research.
Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three DeltaF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Omega.cm(2). In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1beta, TNF-alpha, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development.
Fulcher
M L
ML
Univ. of North Carolina Cystic Fibrosis Center, CB 7248, Rm. 4011 Thurston-Bowles Bldg., Chapel Hill, NC 27599, USA.
Gabriel
S E
SE
Olsen
J C
JC
Tatreau
J R
JR
Gentzsch
M
M
Livanos
E
E
Saavedra
M T
MT
Salmon
P
P
Randell
S H
SH
D000293:Adolescent; D000328:Adult; D001980:Bronchi / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods*; D002461:Cell Line, Transformed; D016922:Cellular Senescence / Q000502:physiology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D015194:Diffusion Chambers, Culture; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D006720:Homozygote; D006801:Humans; D008297:Male; D008875:Middle Aged; D009687:Nuclear Proteins / Q000378:metabolism; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology*; D013539:Simian virus 40; D019098:Telomerase / Q000235:genetics
C069077:BMI1 protein, human; C505032:CFTR protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D063150:Polycomb Repressive Complex 1; C509186:TERT protein, human; D019098:Telomerase