Biological characteristics of Chinese hamster ovary cells transfected with bovine Prnp.
A normal prion protein (PrPc) is converted to a proteaseresistant isoform by an apparent self-propagating activity in transmissible spongiform encephalopathy, a neurodegenerative disease. The cDNA encoding open reading frame (ORF) of the bovine prion protein gene (Prnp) was cloned from Korean cattle by PCR, and was transfected into Chinese hamster ovary (CHO-K1) cells using lipofectamine. The gene expression of the cloned cDNA was confirmed by RT-PCR and Western blotting with the monoclonal antibody, 6H4. Cellular changes in the transfected CHO-K1 cells were investigated using parameters such as MTT, lactate dehydrogenase (LDH), and superoxide dismutase (SOD) activities, as well as nitric oxide (NO) production, and an apoptosis assay. In the MTT and LDH assays, the bovine PrnP-transfectant showed a lower proliferation rate than the wild-type (p < 0.05). Production of NO, after LPS or ConA stimulation, was not detected in either transfectants or CHO-K1 cells. In SOD assay under ConA stimulation, the SOD activity of transfectants was 10 times higher than that of CHO-K1 cells at 6 h after treatment (p < 0.05). The genomic DNA of both the transfectants and control cells began to be fragmented at 6 h after treatment with cyclohexamide. Caspase-3 activity was reduced by transfection with the bovine Prnp (p < 0.05). Conclusively, the viability of transfectants expressing exogenous bovine Prnp was decreased while the capacities for cellular protection against antioxidative stress and apoptosis were increased.
Kang
Sang-Gyun
SG
Department of Infectious Diseases, KRF Zoonotic Disease Priority Research Institute and BK21 Program for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea.
Lee
Deog-Yong
DY
Kang
Mi Lan
ML
Yoo
Han Sang
HS
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D016466:CHO Cells / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D053148:Caspase 3 / Q000378:metabolism; D002417:Cattle; D048708:Cell Growth Processes / Q000502:physiology; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D016643:Encephalopathy, Bovine Spongiform / Q000235:genetics / Q000473:pathology*; D005562:Formazans; D006836:Hydro-Lyases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D011328:Prions / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D013482:Superoxide Dismutase / Q000378:metabolism; D013778:Tetrazolium Salts; D014162:Transfection
D005562:Formazans; D011328:Prions; D013778:Tetrazolium Salts; C079782:MTT formazan; D009569:Nitric Oxide; D013482:Superoxide Dismutase; D053148:Caspase 3; D006836:Hydro-Lyases; C045581:lactate dehydratase