1 Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
2 Laboratory of Immunobiology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-hiroshima, Hiroshima 739-8528, Japan
3 Division of Food Additives, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
4 Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
Correspondence
Yutaka Kikuchi
kikuchi{at}nihs.go.jp
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ABSTRACT |
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INTRODUCTION |
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Several animal cell lines, including mouse neuroblastoma cells (Butler et al., 1988; Race et al., 1987
), mouse hypothalamic neuronal cells (Nishida et al., 2000
; Schätzl et al., 1997
), mouse Schwann cells (Follet et al., 2002
) and rat pheochromocytoma cells (Rubenstein et al., 1984
), have been infected successfully with scrapie agents, and a human neuroblastoma cell line can also be infected with CJD agents (Ladogana et al., 1995
). These cells have been used to study the conversion mechanisms (Lehmann & Harris, 1997
) and the subcellular localization (Naslavsky et al., 1997
; Vey et al., 1996
) of PrPres and to evaluate therapeutic agents (Caughey & Raymond, 1993
; Doh-Ura et al., 2000
). However, the efficiencies of infection and propagation of PrPres are relatively low. The mouse cell line SMB was established from a scrapie-infected mouse brain (Clarke & Haig, 1970
) and has been used to study the properties of PrP (Birkett et al., 2001
). Recently, stable cell lines were established from mouse peripheral neuroglial cells expressing ovine PrP and simian virus 40 T antigen. These cells were readily infectible by sheep PrPSc, a scrapie isoform of PrP (Archer et al., 2004
). However, there are currently no human cell lines that have been used to study the conversion mechanism from PrPC into PrPres.
PrP mRNA is expressed not only in neurons, but also in glia (Moser et al., 1995) and PrPSc accumulates in the cytosol and cell-surface membrane of glial cells (van Keulen et al., 1995
). The role of glial cells in prion disease is not clear. Human glioblastoma T98G cells, like normal cells, become arrested in G1 phase under stationary-phase conditions (Stein, 1979
). In a previous study, we showed that T98G cells express PrPC mRNA constitutively and produce a high level of endogenous PrPC in G1 phase (Kikuchi et al., 2002
). In the present study, we have investigated whether PrPC is converted into PrPres, a marker for prion diseases, in cultured T98G cells under various conditions.
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METHODS |
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Preparation of antibodies.
The preparation of chicken mAb HUC2-13 (IgG) against human PrP peptide residues 2549 was reported previously (Matsuda et al., 1999). The preparation of rabbit polyclonal antibody HPC2 (IgG) against human PrP peptide residues 214230 was also reported previously (Kikuchi et al., 2002
).
Cell culture.
Human glioblastoma cell line T98G (JCRB9041) at nominal passage level 433 was provided by the Japanese Cancer Research Resources Bank (Tokyo, Japan). Human astrocytoma U373MG cells were kindly provided by Dr T. Kasahara (Kyoritsu College of Pharmacy, Tokyo, Japan). Cell cultures stored in liquid nitrogen were thawed as passage 0 (P0) and cultured at 37 °C in monolayers on a T75 plastic tissue-culture flask in RPMI 1640 medium supplemented with 10 % (v/v) heat-inactivated FCS, 60 µg kanamycin ml1 and 10 mM HEPES/NaOH, pH 7·2. All cell lines were subcultivated routinely at a 1 : 5 or 1 : 10 split ratio once a week.
PCR direct sequencing and RFLP analysis.
Extraction of total RNA from the cells and RT-PCR analysis were performed according to a published method (Kikuchi et al., 2002) with slight modifications. Briefly, 5 µg total RNA was treated with DNase I for 15 min at room temperature. Random primers and SuperScript II reverse transcriptase were added to 20 µl (2·5 µg total RNA) and the mixture was incubated at 42 °C for 60 min to synthesize cDNA. Subsequently, 10 µl cDNA solution was subjected to PCR in a total volume of 50 µl, which included 0·2 mM dNTPs, 1 mM MgSO4, 1 U KOD-Plus-DNA polymerase and 50 pmol sense and antisense primers. The amplification programme was as follows: denaturation at 94 °C for 20 s, annealing at 60 °C for 30 s and elongation at 68 °C for 60 s for 40 cycles. Final elongation was performed at 68 °C for 1 min. PCR was carried out in a GeneAmp PCR system 2400 (Applied Biosystems). PCR direct sequencing was performed with a CEQ 2000XL DNA Analysis system (Beckman Coulter) using the primer set for human PrP CDS and an internal primer. Codon 129 polymorphisms were detected by RFLP analysis; the PCR product (200 ng DNA) was digested with 5 U BsaAI for 60 min at 37 °C; after incubation for 20 min at 80 °C, restriction fragments were separated by electrophoresis in 2 % agarose gels and visualized following ethidium bromide staining.
Preparation of whole-cell lysates.
All cell lines were plated at 5·0x105 cells per 9 cm dish (55 cm2) in 10 ml medium on day 0 (D0). The medium was changed every 4 days. At the indicated times, cells were washed twice with ice-cold PBS and scraped into lysis buffer [1·8x104 cells µl1; 10 mM Tris/HCl (pH 7·5), 150 mM NaCl, 1 % sodium deoxycholate, 0·1 % SDS, 1 % NP-40, 10 mM NaF, 1 mM EDTA, 0·5 mM Na3VO3, 10 mM tetrasodium pyrophosphate] with protease inhibitor cocktail [0·06 trypsin inhibitor units (TIU) aprotinin ml1, 20 µM leupeptin and 1 mM PMSF]. After sonication, insoluble material was pelleted by centrifugation at 500 g for 15 min at 4 °C to yield whole-cell lysates. Protein concentration was determined by the BCA protein assay.
Subcellular fractionation.
At the indicated times, cells were washed twice with ice-cold PBS and scraped into PBS/2·5 mM EDTA with the protease inhibitor cocktail. After sonication, insoluble material was pelleted by centrifugation at 500 g for 15 min at 4 °C to yield homogenates. The postnuclear fraction was centrifuged at 100 000 g for 60 min at 4 °C to obtain a cytosolic fraction and a membrane fraction. The membrane fraction was dissolved in PBS/2·5 mM EDTA with the protease inhibitor cocktail. Protein concentration was determined by the BCA protein assay.
Detergent solubility test.
A detergent solubility test was carried out according to a described method (Capellari et al., 2000) with slight modifications. Cells were washed twice with ice-cold PBS and scraped into PBS/2·5 mM EDTA with the protease inhibitor cocktail. After sonication, insoluble material was pelleted by centrifugation at 500 g for 15 min at 4 °C to yield homogenates. The postnuclear fraction was dissolved in 9 vols 0·5 % NP-40/0·5 % deoxycholate/PBS with the protease inhibitor cocktail and centrifuged at 100 000 g for 60 min at 4 °C to obtain a detergent-insoluble pellet fraction and a soluble supernatant fraction. The supernatant fraction was precipitated with 4 vols methanol for 16 h at 20 °C. Both fractions were resuspended in the same volume of lysis buffer.
Protease-resistant PrP assay.
To generate material for the protease-resistant PrP assay, aliquots of the sample (50 µg protein) were precipitated with 4 vols methanol for 16 h at 20 °C to remove the protease inhibitor cocktail (Capellari et al., 2000), centrifuged at 14 000 g for 15 min at 4 °C and the pellet was dissolved in 50 mM Tris/HCl (pH 7·2). Samples were treated with PK (at 10 µg ml1 unless stated otherwise) at 37 °C for 30 min, according to a described method (Caughey et al., 1999
). After incubation, digestion was stopped by the addition of AEBSF to 4 mM. Samples were prepared with the protease inhibitor cocktail at a concentration that did not inhibit the activity of PK (Fig. 1a
, lane 1).
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Immunoblotting.
Usually, 50 µg total protein (prepared from approximately 1·7x105 cells) was subjected to SDS gel electrophoresis. Briefly, aliquots of the samples were mixed with 2x electrophoresis sample buffer. After boiling for 10 min, the samples were electrophoresed on 12·5 % acrylamide gel and the proteins were transferred onto PVDF membranes. The membranes were blocked with 0·5 % casein in PBS (casein/PBS) and incubated with anti-prion antibodies in casein/PBS. Immunoreactive bands were visualized with HRP-conjugated anti-IgG and SuperSignal West Femto Maximum Sensitivity substrate, according to the manufacturer's instructions (Pierce Biotechnology).
Indirect immunofluorescence staining.
T98G cell monolayers grown on a 15 mm glass coverslip (Matsunami) in a 9 cm dish (55 cm2) were maintained in 10 ml medium. At the indicated times, cells were washed twice with ice-cold PBS and then fixed with 3·7 % formaldehyde in PBS for 30 min at 4 °C. The fixed cells were washed twice with PBS and then treated with 0·2 % Triton X-100 in PBS for 15 min at room temperature. The cells were blocked with 10 % normal goat serum in PBS (NGS/PBS) for 60 min and incubated with antibody (100 ng ml1) for 16 h at 4 °C. After extensive washing with 0·05 % Tween 20/PBS, cells were treated with Alexa 594 goat anti-mouse IgG (H+L) conjugate (5 µg ml1) (Molecular Probes) in NGS/PBS for 1 h at 4 °C, washed with 0·05 % Tween 20/PBS and mounted with 2·5 % DABCO/90 % glycerin/PBS. The stained cells were observed and photographed with the aid of a fluorescence microscope (Olympus).
Competitive ELISA.
ELISA was carried out according to a method described previously (Kikuchi et al., 1991). For a dilution buffer, casein/PBS was used throughout the present study. Briefly, the wells were coated with 100 ng recombinant bovine PrP (rBoPrP) (Takekida et al., 2002
) in PBS and left at 4 °C overnight. Appropriately diluted standard rBoPrP solutions or samples were added to the antigen-coated wells and incubated at room temperature for 60 min, in a total volume of 50 µl, with 6H4 antibody (460 pg). The wells were washed, incubated with
-galactosidase-conjugated goat anti-mouse IgG for 60 min, washed again and then incubated with 4-MUG as a substrate at 37 °C for 60 min. Enzyme activity was determined by fluorescence intensity measurements.
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RESULTS |
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Examination of phenotypic variants of PrPres
We first asked whether an inherited or a sporadic CJD-like form of PrPres was propagated in T98G cells. Inherited prion diseases are determined by mutations in the 762 bp CDS of the prion protein gene (PRNP) (Kovács et al., 2002). We performed PCR direct sequencing of the PRNP mRNA that was expressed in short- and long-term cultured T98G cells and found no mutations other than the presence of both adenine and guanine at the first position of codon 129 (the basis of the common M129V polymorphism) (data not shown). When digested by BsaAI, the 806 bp PCR product from the M129V haplotype (Fig. 2a
, lane 1) yielded products of 402 and 404 bp and also undigested wild-type product (Fig. 2a
, lane 2), which we confirmed by RFLP analysis. These results indicated that T98G cells were heterozygotes, having both methionine and valine at codon 129 of PRNP with no coding-region mutation.
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Confirming heterogeneity of PrPres by immunoblotting with sets of anti-PrP antibodies
To further investigate the heterogeneity of PrPres from long-term cultured T98G cells, we determined the antigenicity of PrPres. By immunoblotting with sets of antibodies to PrP (Kikuchi et al., 2002), we detected a full-length PrP (35 kDa) in lysates from P40D40 T98G cells that reacted with the anti-N terminus PrP antibody HUC2-13 (Fig. 3a
, lane 2), as well as with the 6H4 antibody (Fig. 3c
, lane 2). Following PK treatment of the lysates, the 31 kDa band was still detected by 6H4 antibody (Fig. 3c
, lane 1), but not by HUC2-13 antibody (Fig. 3a
, lane 1), indicating that PK treatment had cleaved the N terminus of PrPres. The 31 kDa band was also detected by the anti-C terminus PrP antibody HPC2 (Fig. 3d
, lane 1). HPC2 antibody, which reacts strongly with the deglycosylated form of PrPC, but weakly with the glycosylated form (Kikuchi et al., 2002
), also recognized the N-terminally truncated form of PrPres. Surprisingly, the 3F4 antibody, which recognizes residues 109112, failed to detect the N-terminally truncated form of PrPres (Fig. 3b
), such as is seen with the HUC2-13 antibody (Fig. 3a
). These experiments showed that the N-terminally truncated form of PrPres in T98G cells lacks the epitope that is recognized by the 3F4 antibody.
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DISCUSSION |
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Direct sequencing of amplified PRNP mRNA and RFLP analysis indicated that the T98G cells were heterozygotes at codon 129 (129M/V) and that no new coding mutations were present in cells that had been subjected to long-term cultures. The deglycosylated form of PK-treated PrPres in T98G cells migrated at approximately 18 kDa. In human prion diseases, two major types of PrPres can be identified, based on electrophoretic migration; the relative molecular mass of the unglycosylated form is approximately 21 kDa (described as type 1) or 19 kDa (described as type 2) (Parchi et al., 1997). Accordingly, PrPres in T98G cells is similar to the previously described MV2 phenotypic variant (Parchi et al., 1999a
). However, the size of the deglycosylated PK-resistant fragment in T98G cells was smaller than that of the corresponding fragments observed in type 2 PrPres. Most importantly, the 3F4 antibody, which is a well-characterized antibody known to target residues 109112 as its epitope (Kascsak et al., 1987
; Matsunaga et al., 2001
), did not react with PK-digested PrPres in T98G cells, suggesting that the N-terminal PrP region up to residue 109 might be absent in PK-treated PrPres in T98G cells. Human PrPres peptide is divided into three regions that are defined by their PK-cleavage patterns: an N-terminal region (residues 2373) that is invariably PK-sensitive, a C-terminal region (residues 103231) that is invariably PK-resistant and a variably digested region (residues 74102), where the major cleavage sites are at G82 in type 1 and at S97 in type 2 (Parchi et al., 2000
). The 3F4 antibody was used to type PrPres (Parchi et al., 2000
). Therefore, there are striking differences in the antigenicity, which reflect the PK-cleavage patterns, between type 2 PrPres in sporadic CJD brain and in T98G cells. It is unlikely, but not impossible, that PK treatment generated conformational changes in the mid-region of PrPres that interfered with epitope recognition by the 3F4 antibody. Further studies are needed to classify the type of PrPres in lysates from long-term cultured T98G cells.
So far, human PrPSc has been analysed on immunoblots with the 3F4 antibody. Our finding may explain why previous studies have failed to detect PrPres in cultured cells. Interestingly, an N-terminally truncated 18 kDa fragment of PrP (designated C1) in normal and sporadic CJD brains has similar properties except that it is PK-sensitive; it is recognized by the anti-C terminus antibody, but not by the 3F4 antibody, is cleaved around residue 111 and is associated with cell membranes (Chen et al., 1995). PrPC from human brain homogenates (n=6) originally displayed a partial PK resistance (20 µg ml1 for 10 min) and has been detected by the antibody that recognizes residues 145163, but not by the 3F4 antibody (Buschmann et al., 1998
). Taking the data from the various studies of PrP immunoreactivity into consideration, we believe that it would be better to incorporate an additional antibody that recognizes the C terminus of PrP into the standardly used protease resistance-dependent PrPSc assay.
Among the sets of antibodies used in this study, the anti-N-terminal portion antibodies (HUC2-13 and 3F4) reacted strongly with the fully glycosylated form and moderately with the partially glycosylated form. In contrast, the antibodies against the C-terminal portion of PrP (6H4 and HPC) reacted moderately with the fully glycosylated form and strongly with the partially glycosylated form. It is possible that PK digestion induces a conformational change of digested PrP and enhances its immunoreactivity to the anti-C-terminal antibodies. Recently, it has been reported that the amino acid motif Tyr-Tyr-Arg (YYR), located in a -sheet, is exposed in PrPSc, whilst it is cryptic in PrPC, and that antibodies recognize YYR in PrPSc, but not in PrPC (Paramithiotis et al., 2003
). Another paper has reported that PK digestion enhances immunoreactivity to the anti-PrP antibody that recognizes the epitope YYR, located in a
-sheet (Brun et al., 2004
). These reports suggest that conformation of the C-terminal portion of PrPSc is essential for immunoreactivity of anti-YYR antibodies. The 6H4 antibody also recognizes residues 144152 of PrP, including a YYR motif that is located in an
-helix, not in a
-sheet (Korth et al., 1997
). Further study is needed to clarify the immunoreactivity of anti-C-terminal PrP antibodies.
It has been proposed that PrPC is converted into PrPres either on the cell surface or in endocytic cellular compartments. PrPC is a surface protein that contains a glycosylphosphatidylinositol anchor (Stahl et al., 1987). A portion of PrPSc is also localized on the cell surface of scrapie-infected mouse neuroblastoma ScN2a cells (Naslavsky et al., 1997
; Vey et al., 1996
), although it is also found in lysosomes (Taraboulos et al., 1990
). Subcellular localization of PrPres in long-term cultured T98G cells was similar to that of PrPSc-infected cells, being present on the cell surface. PrPSc in ScN2a cells is sedimented by centrifugation in non-ionic detergents (Caughey et al., 1991
). Mutant PrP in stably transfected Chinese hamster ovary cells, which express murine homologues associated with human inherited prion diseases, is also non-ionic detergent-insoluble (Lehmann & Harris, 1996
). However, the PrPres in T98G cells is detergent-soluble. PrPres in the human neuroblastoma cell line M-17 BE(2)C carrying the familial subtype CJD, the glutamic acid to lysine substitution at codon 200 (E200K), is also partially non-ionic detergent-insoluble (Capellari et al., 2000
). The present study indicates that not all PrPres is non-ionic detergent-insoluble.
Many cultured cells that express PrPres mutants carrying substitutions of inherited prion disease show considerably less protease resistance (up to 3·3 µg ml1 for 10 min), compared with PrPres mutants isolated from the human brain (Capellari et al., 2000; Harris, 2001
). In contrast, the PrPres in T98G cells displayed a high resistance to digestion with PK (10 µg ml1 for 30 min), but was less resistant than PrPres in brain homogenates of sporadic CJD (up to 100 µg ml1 for 24 h). Sporadic CJD is typically characterized by widespread spongiform degeneration with loss of neurons, gliosis and formation of amyloid plaques (Parchi et al., 1999a
). It has recently been reported that six cases of sporadic fatal insomnia, a prion disease mimicking fatal familial insomnia, had no coding-region mutation of PRNP with the 129 M/M genotype and an approximately 19 kDa deglycosylated PrPres, the same as that of type 2 (Mastrianni et al., 1999
; Parchi et al., 1999b
). Familial progressive subcortical gliosis may also be a prion disease, characterized by astrogliosis at the cortexwhite matter junction (Petersen et al., 1995
). All patients from two families with that disease showed no coding-region mutation of PRNP, the 129 M/M genotype and the 18·119·3 kDa form of deglycosylated PrPres (Petersen et al., 1995
). T98G cells were grown out of human glioblastoma multiforma tumour tissue of a 61-year-old Caucasian man (Stein, 1979
). We consider it possible that he also had a sporadic form of prion disease.
Conversion from PrPC into PrPres is an important process, because most prion diseases are characterized by presence of PrPres. Some knowledge of the conversion mechanism is based on studies of scrapie-infected cells. Recently, it has been reported that several conditions can induce the formation of PrPres in cultured cells. Proteasome inhibitors cause accumulation of the unglycosylated form of PrPres in treated cells (Lehmann & Harris, 1997; Ma & Lindquist, 1999
; Yedidia et al., 2001
). PrP that misfolds during maturation in the endoplasmic reticulum is delivered to the cytosol for degradation by proteasomes (Béranger et al., 2002
; Ma & Lindquist, 2001
; Yedidia et al., 2001
). It has been hypothesized the conversion into PrPres might occur when the number of PrP molecules exceeds the capacity of the cell to degrade them (Ma & Lindquist, 2002
). Another study showed that manganese-treated mouse astrocytes express the glycosylated form of PrPres (Brown et al., 2000
). Here, we report for the first time the conversion of PrPC into PrPres in the widely used human glioblastoma cell line T98G; a large number of passages and prolonged incubation under routine cell-culture conditions are required. In vitro-generated PrPres is reportedly not sufficient for the production of infectivity (Caughey et al., 2001
; Hill et al., 1999
) and further study is needed to clarify the infectivity of PrPres in T98G cells (indeed, caution should be taken with T98G cells in the laboratory). Infectivity assays of PrPres in T98G cells are now in progress in transgenic mice.
In conclusion, T98G cells should be a useful model for studying the mechanisms of PrPC conversion into PrPres.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Béranger, F., Mangé, A., Goud, B. & Lehmann, S. (2002). Stimulation of PrPC retrograde transport toward the endoplasmic reticulum increases accumulation of PrPSc in prion-infected cells. J Biol Chem 277, 3897238977.
Birkett, C. R., Hennion, R. M., Bembridge, D. A., Clarke, M. C., Chree, A., Bruce, M. E. & Bostock, C. J. (2001). Scrapie strains maintain biological phenotypes on propagation in a cell line in culture. EMBO J 20, 33513358.
Brown, D. R., Hafiz, F., Glasssmith, L. L., Wong, B.-S., Jones, I. M., Clive, C. & Haswell, S. J. (2000). Consequences of manganese replacement of copper for prion protein function and proteinase resistance. EMBO J 19, 11801186.
Brun, A., Castilla, J., Ramírez, M. A. & 8 other authors (2004). Proteinase K enhanced immunoreactivity of the prion protein-specific monoclonal antibody 2A11. Neurosci Res 48, 7583.[CrossRef][Medline]
Buschmann, A., Kuczius, T., Bodemer, W. & Groschup, M. H. (1998). Cellular prion proteins of mammalian species display an intrinsic partial proteinase K resistance. Biochem Biophys Res Commun 253, 693702.[CrossRef][Medline]
Butler, D. A., Scott, M. R. D., Bockman, J. M., Borchelt, D. R., Taraboulos, A., Hsiao, K. K., Kingsbury, D. T. & Prusiner, S. B. (1988). Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins. J Virol 62, 15581564.[Medline]
Capellari, S., Parchi, P., Russo, C. M., Sanford, J., Sy, M. S., Gambetti, P. & Petersen, R. B. (2000). Effect of the E200K mutation on prion protein metabolism. Comparative study of a cell model and human brain. Am J Pathol 157, 613622.
Caughey, B. & Raymond, G. J. (1993). Sulfated polyanion inhibition of scrapie-associated PrP accumulation in cultured cells. J Virol 67, 643650.[Abstract]
Caughey, B., Raymond, G. J., Ernst, D. & Race, R. E. (1991). N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state. J Virol 65, 65976603.[Medline]
Caughey, B., Horiuchi, M., Demaimay, R. & Raymond, G. J. (1999). Assays of protease-resistant prion protein and its formation. Methods Enzymol 309, 122133.[Medline]
Caughey, B., Raymond, G. J., Callahan, M. A., Wong, C., Baron, G. S. & Xiong, L.-W. (2001). Interactions and conversions of prion protein isoforms. Adv Protein Chem 57, 139169.[CrossRef][Medline]
Chen, S. G., Teplow, D. B., Parchi, P., Teller, J. K., Gambetti, P. & Autilio-Gambetti, L. (1995). Truncated forms of the human prion protein in normal brain and in prion diseases. J Biol Chem 270, 1917319180.
Clarke, M. C. & Haig, D. A. (1970). Evidence for the multiplication of scrapie agent in cell culture. Nature 225, 100101.[Medline]
Doh-Ura, K., Iwaki, T. & Caughey, B. (2000). Lysosomotropic agents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation. J Virol 74, 48944897.
Follet, J., Lemaire-Vieille, C., Blanquet-Grossard, F. & 8 other authors (2002). PrP expression and replication by Schwann cells: implications in prion spreading. J Virol 76, 24342439.
Harris, D. A. (2001). Biosynthesis and cellular processing of the prion protein. Adv Protein Chem 57, 203228.[Medline]
Hill, A. F., Antoniou, M. & Collinge, J. (1999). Protease-resistant prion protein produced in vitro lacks detectable infectivity. J Gen Virol 80, 1114.[Abstract]
Kascsak, R. J., Rubenstein, R., Merz, P. A., Tonna-DeMasi, M., Fersko, R., Carp, R. I., Wisniewski, H. M. & Diringer, H. (1987). Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. J Virol 61, 36883693.[Medline]
Kikuchi, Y., Irie, M., Yoshimatsu, K. & 8 other authors (1991). A monoclonal antibody to scopolamine and its use for competitive enzyme-linked immunosorbent assay. Phytochemistry 30, 32733276.[CrossRef][Medline]
Kikuchi, Y., Kakeya, T., Yamazaki, T. & 7 other authors (2002). G1-dependent prion protein expression in human glioblastoma cell line T98G. Biol Pharm Bull 25, 728733.[CrossRef][Medline]
Korth, C., Stierli, B., Streit, P. & 14 other authors (1997). Prion (PrPSc)-specific epitope defined by a monoclonal antibody. Nature 390, 7477.[CrossRef][Medline]
Kovács, G. G., Trabattoni, G., Hainfellner, J. A., Ironside, J. W., Knight, R. S. G. & Budka, H. (2002). Mutations of the prion protein gene phenotypic spectrum. J Neurol 249, 15671582.[Medline]
Ladogana, A., Liu, Q., Xi, Y. G. & Pocchiari, M. (1995). Proteinase-resistant protein in human neuroblastoma cells infected with brain material from Creutzfeldt-Jakob patient. Lancet 345, 594595.
Lehmann, S. & Harris, D. A. (1996). Mutant and infectious prion proteins display common biochemical properties in cultured cells. J Biol Chem 271, 16331637.
Lehmann, S. & Harris, D. A. (1997). Blockade of glycosylation promotes acquisition of scrapie-like properties by the prion protein in cultured cells. J Biol Chem 272, 2147921487.
Ma, J. & Lindquist, S. (1999). De novo generation of a PrPSc-like conformation in living cells. Nat Cell Biol 1, 358361.[CrossRef][Medline]
Ma, J. & Lindquist, S. (2001). Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation. Proc Natl Acad Sci U S A 98, 1495514960.
Ma, J. & Lindquist, S. (2002). Conversion of PrP to a self-perpetuating PrPSc-like conformation in the cytosol. Science 298, 17851788.
Mastrianni, J. A., Nixon, R., Layzer, R., Telling, G. C., Han, D., DeArmond, S. J. & Prusiner, S. B. (1999). Prion protein conformation in a patient with sporadic fatal insomnia. N Engl J Med 340, 16301638.
Matsuda, H., Mitsuda, H., Nakamura, N., Furusawa, S., Mohri, S. & Kitamoto, T. (1999). A chicken monoclonal antibody with specificity for the N-terminal of human prion protein. FEMS Immunol Med Microbiol 23, 189194.[CrossRef][Medline]
Matsunaga, Y., Peretz, D., Williamson, A., Burton, D., Mehlhorn, I., Groth, D., Cohen, F. E., Prusiner, S. B. & Baldwin, M. A. (2001). Cryptic epitopes in N-terminally truncated prion protein are exposed in the full-length molecule: dependence of conformation on pH. Proteins 44, 110118.[CrossRef][Medline]
Moser, M., Colello, R. J., Pott, U. & Oesch, B. (1995). Developmental expression of the prion protein gene in glial cells. Neuron 14, 509517.[Medline]
Naslavsky, N., Stein, R., Yanai, A., Friedlander, G. & Taraboulos, A. (1997). Characterization of detergent-insoluble complexes containing the cellular prion protein and its scrapie isoform. J Biol Chem 272, 63246331.
Nishida, N., Harris, D. A., Vilette, D., Laude, H., Frobert, Y., Grassi, J., Casanova, D., Milhavet, O. & Lehmann, S. (2000). Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein. J Virol 74, 320325.
Paramithiotis, E., Pinard, M., Lawton, T. & 19 other authors (2003). A prion protein epitope selective for the pathologically misfolded conformation. Nat Med 9, 893899.[CrossRef][Medline]
Parchi, P., Capellari, S., Chen, S. G. & 8 other authors (1997). Typing prion isoforms. Nature 386, 232234.[CrossRef][Medline]
Parchi, P., Giese, A., Capellari, S. & 15 other authors (1999a). Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol 46, 224233.[CrossRef][Medline]
Parchi, P., Capellari, S., Chin, S. & 7 other authors (1999b). A subtype of sporadic prion disease mimicking fatal familial insomnia. Neurology 52, 17571763.
Parchi, P., Zou, W., Wang, W. & 10 other authors (2000). Genetic influence on the structural variations of the abnormal prion protein. Proc Natl Acad Sci U S A 97, 1016810172.
Petersen, R. B., Tabaton, M., Chen, S. G. & 10 other authors (1995). Familial progressive subcortical gliosis: presence of prions and linkage to chromosome 17. Neurology 45, 10621067.[Abstract]
Prusiner, S. B. (2001). Prions. In Fields Virology, 4th edn, pp. 30633087. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Race, R. E., Fadness, L. H. & Chesebro, B. (1987). Characterization of scrapie infection in mouse neuroblastoma cells. J Gen Virol 68, 13911399.[Abstract]
Rubenstein, R., Carp, R. I. & Callahan, S. M. (1984). In vitro replication of scrapie agent in a neuronal model: infection of PC12 cells. J Gen Virol 65, 21912198.[Abstract]
Satoh, J., Kurohara, K., Yukitake, M. & Kuroda, Y. (1998). Constitutive and cytokine-inducible expression of prion protein gene in human neural cell lines. J Neuropathol Exp Neurol 57, 131139.[Medline]
Schätzl, H. M., Laszlo, L., Holtzman, D. M., Tatzelt, J., DeArmond, S. J., Weiner, R. I., Mobley, W. C. & Prusiner, S. B. (1997). A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis. J Virol 71, 88218831.[Abstract]
Stahl, N., Borchelt, D. R., Hsiao, K. & Prusiner, S. B. (1987). Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51, 229240.[Medline]
Stein, G. H. (1979). T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro. J Cell Physiol 99, 4354.[Medline]
Takekida, K., Kikuchi, Y., Yamazaki, T. & 7 other authors (2002). Quantitative analysis of prion protein by immunoblotting. J Health Sci 48, 288291.[CrossRef]
Taraboulos, A., Serban, D. & Prusiner, S. B. (1990). Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells. J Cell Biol 110, 21172132.[Abstract]
van Keulen, L. J., Schreuder, B. E., Meloen, R. H., Poelen-van den Berg, M., Mooij-Harkes, G., Vromans, M. E. & Langeveld, J. P. (1995). Immunohistochemical detection and localization of prion protein in brain tissue of sheep with natural scrapie. Vet Pathol 32, 299308.[Abstract]
Vey, M., Pilkuhn, S., Wille, H., Nixon, R., DeArmond, S. J., Smart, E. J., Anderson, R. G. W., Taraboulos, A. & Prusiner, S. B. (1996). Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains. Proc Natl Acad Sci U S A 93, 1494514949.
Yedidia, Y., Horonchik, L., Tzaban, S., Yanai, A. & Taraboulos, A. (2001). Proteasomes and ubiquitin are involved in the turnover of the wild-type prion protein. EMBO J 20, 53835391.
Received 18 February 2004;
accepted 19 July 2004.
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