NR AXTK
AU Nakamitsu,S.; Kurokawa,A.; Uryu,M.; Horiuchi,M.
TI Cell-Density-Dependent Fluctuation of PrP-res in Prion-Infected Neuro2a Cells
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.10
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB Cells persistently infected with prion are widely used for researches in prion diseases such as studying the mechanism of prion propagation and screening anti-prion compounds. Protease-resistant PrP, designated PrP-res, is usually detected in cells infected with prion, and the detection of PrP-res is often used as an indicator of the existence of prion. The amount of PrP-res in prion-infected cells is expected to fluctuate depending on culture conditions. Therefore, we investigated the fluctuation of PrP-res and total PrP in Neuro2a (N2a) cells infected with prion. N2a sublines, Sc2a3 and N2a5, and N2aII, mouse PrPc overexpressing N2a subline, were used. These sublines persistently infected with Chandler strain (ScN2a3, ScN2a5, and ScN2aII) were seeded at various cell numbers, and cells were harvested every 24 hr for the detection of total PrP and PrP-res. The amount of PrP-res per unit showed timedependent increase, especially the level of PrP-res markedly increased when cells grew to sub-confluent and then confluent. Total PrP levels in ScN2a3 and ScN2a5 also showed cell-density-dependent increase. Compared with an immediately before passage, the PrP-res level was obviously low at 24 hours after passage. Further investigation revealed that the decrease of PrP-res level within 24 hr after passage was not due to the digestion of PrP-res with trypsin at the passage, but the level of PrP-res decreased rapidly within 24 hours after seeding the cells. These results suggested that PrP-res levels in ScN2a sublines were fluctuated by physiological conditions determined by cell density and/or cell growth. To investigate the effect of cell density on PrP-res formation, ScN2a3 and ScN2a5 were co-cultured with N2a1 subline that is resistant to Chandler strain. The PrP-res levels in ScN2a3 and ScN2a5 increased in proportion to the increase of co-cultured N2a1 cell numbers. In contrast, conditioned medium obtained from N2a and ScN2a sublines did not have major effect on the level of PrP-res in the corresponding ScN2a sublines. Taken together, these results suggest that certain cellular microenvironment associated with cell density, possibly direct contact between cells, will be involved in the PrP-res formation in ScN2a cells.
AD S. Nakamitsu, A. Kurokawa, M. Uryu, M. Horiuchi, Graduate School of Veterinary Medicine, Hokkaido University, Japan
SP englisch
PO Schottland