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G1-dependent prion protein expression in human glioblastoma cell line T98G
Yutaka Kikuchi1, Tomoshi Kakeya, Takeshi Yamazaki
1Division of Microbiology, National Institute of Health Sciences, Tokyo, Japan. kikuchi@nihs.go.jp
Abstract:
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.
Insights
Human glioblastoma cells (T98G) produce cellular prion protein (PrP(C)), with production levels influenced by cell cycle progression. This suggests a link between PrP(C) expression and cell division phases in cancer cells.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Cellular prion protein (PrP(C)) is implicated in various cellular functions and diseases.
- Glioblastoma is an aggressive form of brain cancer with complex cellular mechanisms.
- Understanding PrP(C) expression in cancer cells can offer insights into disease progression.
Purpose of the Study:
- To investigate the production and regulation of cellular prion protein (PrP(C)) in the human glioblastoma cell line T98G.
- To determine if PrP(C) expression is influenced by cell cycle or culture conditions.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) to confirm PrP mRNA expression.
- Immunoblot analysis using anti-PrP antibody 3F4 to detect PrP(C) protein.
- Treatment with tunicamycin and N-glycosidase F to analyze glycosylation.
- Cell culture under varying time points, cell densities, and serum conditions.
Main Results:
- T98G cells express PrP mRNA and produce PrP(C) protein.
- PrP(C) exists in glycosylated (35 kDa) and deglycosylated (25 kDa) forms.
- PrP(C) production increases with prolonged culture and high cell density.
- Serum-free conditions at low density mimic high-density production.
- PrP(C) production is cell cycle-dependent, likely in the G1 phase.
Conclusions:
- PrP(C) is actively produced by T98G glioblastoma cells.
- PrP(C) production is modulated by cell cycle progression, particularly the G1 phase.
- These findings contribute to understanding the role of PrP(C) in glioblastoma biology.