Development and characterization of conditionally immortalized gastric epithelial cell lines from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene.
Conditionally immortalized gastric epithelial cell lines were established from transgenic rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene. Gastric mucosal cells and epithelial tissues isolated from the stomach of the transgenic rats were cultured at permissive temperature (33 degrees C), and proliferative cells were cloned by colony formation. Six cell lines (designated as RGE1-01, RGE1-02, RGE1-03, RGE1-21, RGE1-22 and RGE2-01) showing epithelial-like morphology have been established. All cells grew at 33 degrees C, but did not at nonpermissive temperature (39 degrees C). High expression level of large T-antigen in the nuclei was observed at 33 degrees C, whereas the expression level was gradually decreased in a time-dependent manner at 39 degrees C. These results suggest that the temperature-sensitive growth characteristics arise as a result of a function of the tsSV40 large T-antigen. None of the cell lines were transformed as judged by anchorage-independent growth assay. Immunocytochemical findings indicated that all cells expressed epithelial cell markers including cytoskeletal (cytokeratin and actin), basement membrane (laminin and collagen type IV) and junctional complex (ZO-1 and desmoplakin I+II) proteins at 33 degrees C. All cells expressed mRNA of cathepsin E, a pit cell marker. Moreover, transepithelial resistance was observed between apical and basolateral sides in the cells. RGE1-22 cells produced prostaglandin E(2). Levels of mRNA for cathepsin E, transepithelial resistance and prostaglandin E(2) were influenced by the nonpermissive temperature. Thus, these conditionally immortalized gastric cell lines which preserve some epithelial cell characteristics will provide a useful in vitro model of gastric epithelium.
Molecular Genetics Research Center, Toyama Medical and Pharmaceutical University, Japan. ytabu@ms.toyama-mpu.ac.jp
Yoshiaki
Y
Tabuchi
Yuko
Y
Arai
Shoichiro
S
Ohta
Hiroki
H
Shioya
Ri-ichi
R
Takahashi
Masatsugu
M
Ueda
Noriaki
N
Takeguchi
Shinji
S
Asano
Masuo
M
Obinata
D000818:Animals; D030801:Animals, Genetically Modified; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D015415:Biomarkers; D020059:Cathepsin E / Q000235:genetics; D002455:Cell Division; D002461:Cell Line, Transformed; D015232:Dinoprostone / Q000032:analysis; D017097:Electric Impedance; D004847:Epithelial Cells / Q000166:cytology*; D005753:Gastric Mucosa / Q000166:cytology; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D013539:Simian virus 40 / Q000235:genetics*; D013696:Temperature
D000957:Antigens, Viral, Tumor; D015415:Biomarkers; D012333:RNA, Messenger; D020059:Cathepsin E; D015232:Dinoprostone