Propagation of a protease-resistant form of prion protein in long-term cultured human glioblastoma cell line T98G.
Human prion diseases, such as Creutzfeldt-Jakob disease (CJD), a lethal, neurodegenerative condition, occur in sporadic, genetic and transmitted forms. CJD is associated with the conversion of normal cellular prion protein (PrP(C)) into a protease-resistant isoform (PrP(res)). The mechanism of the conversion has not been studied in human cell cultures, due to the lack of a model system. In this study, such a system has been developed by culturing cell lines. Human glioblastoma cell line T98G had no coding-region mutations of the prion protein gene, which was of the 129 M/V genotype, and expressed endogenous PrP(C) constitutively. T98G cells produced a form of proteinase K (PK)-resistant prion protein fragment following long-term culture and high passage number; its deglycosylated form was approximately 18 kDa. The PK-treated PrP(res) was detected by immunoblotting with the mAb 6H4, which recognizes residues 144-152, and a polyclonal anti-C-terminal antibody, but not by the mAb 3F4, which recognizes residues 109-112, or the anti-N-terminal mAb HUC2-13. These results suggest that PrP(C) was converted into a proteinase-resistant form of PrP(res) in T98G cells.
Kikuchi
Yutaka
Y
Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Kakeya
Tomoshi
T
Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Sakai
Ayako
A
Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Takatori
Kosuke
K
Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Nakamura
Naoto
N
Laboratory of Immunobiology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-hiroshima, Hiroshima 739-8528, Japan.
Matsuda
Haruo
H
Laboratory of Immunobiology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-hiroshima, Hiroshima 739-8528, Japan.
Yamazaki
Takeshi
T
Division of Food Additives, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Tanamoto
Ken-Ichi
KI
Division of Food Additives, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Sawada
Jun-Ichi
JI
Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
D045744:Cell Line, Tumor; D004351:Drug Resistance; D019286:Endopeptidase K / Q000494:pharmacology; D005909:Glioblastoma; D006801:Humans; D015151:Immunoblotting; D042541:Intracellular Space / Q000378:metabolism; D011328:Prions / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism
D011328:Prions; D020033:Protein Isoforms; D019286:Endopeptidase K