title: Biosynthetic and growth abnormalities are associated with high-level expression of CFTR in heterologous cells.
abstract: An inducible gene amplification system was utilized to study the effects of overexpression of cystic fibrosis transmembrane conductance regulator (CFTR) in vitro. BTS, a monkey kidney cell line expressing a temperature-sensitive simian virus 40 (SV-40) large T antigen was stably transfected at the nonpermissive temperature with a plasmid containing an SV-40 origin of replication and the cDNA for either the wild-type CFTR or the mutant G551D-CFTR. Shift of the isolated cell lines to the permissive temperature resulted in induction and accumulation to high levels of the CFTR plasmid, mRNA, and protein. However, high-level expression of CFTR was transient in both BTS-CFTR and BTS-G551D cells due to a decrease in their respective levels of CFTR mRNA. Because G551D-CFTR only exhibits residual Cl channel activity, this suggests that the observed downregulation with BTS-G551D cells may have been induced by either the physical presence of high amounts of CFTR or some low threshold level of Cl- channel activity. Examination of cell growth properties revealed a correlation between high-level expression of wild-type CFTR and growth arrest of the cells at the G2/M phase. However, similar induction of the G551D-CFTR mutant showed only a slight growth inhibition and little enrichment of cells at the G2/M phase. Cytofluorographic analysis further revealed that BTS-CFTR cells were significantly larger than parental BTS or BTS-G551D cells at all stages of the cell cycle. These results indicate that CFTR overexpression is capable of inducing its own downregulation and that high levels of Cl- channel activity can result in increased cell volume and subsequent cell growth abnormalities.
authors.affiliation: Genzyme Corporation, Framingham, Massachusetts 01701-9322, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Schiavi
authors.forename: N
authors.initials: N
authors.lastname: Abdelkader
authors.forename: S
authors.initials: S
authors.lastname: Reber
authors.forename: S
authors.initials: S
authors.lastname: Pennington
authors.forename: R
authors.initials: R
authors.lastname: Narayana
authors.forename: J M
authors.initials: JM
authors.lastname: McPherson
authors.forename: A E
authors.initials: AE
authors.lastname: Smith
authors.forename: H
authors.initials: H
authors.lastname: Hoppe
authors.forename: S H
authors.initials: SH
authors.lastname: Cheng
mesh_terms: D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002468:Cell Physiological Phenomena; D002477:Cells / Q000166:cytology / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D016195:G2 Phase; D000882:Haplorhini; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008938:Mitosis; D009154:Mutation; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator