G1-dependent prion protein expression in human glioblastoma cell line T98G.
Human glioblastoma cell line T98G produced a cellular form of prion protein (PrP(C)), and we confirmed expression of PrP mRNA by RT-PCR. Immunoblot analysis of whole cell lysate revealed one major (35 kDa) and two faint bands (31, 25 kDa) that reacted with monoclonal anti-human PrP antibody 3F4. Cells treated with tunicamycin produced only a 25 kDa band, representing a deglycosylated form of PrP. Similarly, peptide: N-glycosidase F treatment of whole cell lysate altered the Asn-linked form to the deglycosylated form. When T98G cells were cultured for a longer period, the amount of PrP(C) per cell increased on Day 4 to 16 in a time-dependent manner. When the cells were cultured at high cell-density, the cells on Day 4 produced the same amount of PrP(C) as those on Day 16 of the usual culture. Moreover, in a serum-free medium, cells cultured at a low cell-density produced the same amount of PrP(C) as those cultured at the high cell-density. These results demonstrate that PrP(C) production in T98G cells was dependent on the phase of the cell cycle, probably the G1 phase.
Kikuchi
Yutaka
Y
Division of Microbiology, National Institute of Health Sciences, Tokyo, Japan. kikuchi@nihs.go.jp
Kakeya
Tomoshi
T
Yamazaki
Takeshi
T
Takekida
Kaori
K
Nakamura
Naoto
N
Matsuda
Haruo
H
Takatori
Kosuke
K
Tanimura
Akio
A
Tanamoto
Ken-ichi
K
Sawada
Jun-ichi
J
D001932:Brain Neoplasms / Q000378:metabolism*; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis; D017931:DNA Primers; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D009687:Nuclear Proteins / Q000378:metabolism*; D018621:PrPC Proteins / Q000096:biosynthesis*; D000072002:Prion Proteins; D011328:Prions / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D009687:Nuclear Proteins; C512656:PRNP protein, human; D018621:PrPC Proteins; D000072002:Prion Proteins; D011328:Prions; D012313:RNA; D004247:DNA