SAMR1 and SAMP8 mice have been maintained as inbred strains in the Institute for Basic Research animal colony and the Ilsong Institute of Life Science animal colony. Pathogen-free SAMR1, SAMP8, and ICR (Daehan Biolink, Korea) animals have been housed in cages in a clean facility. All animals are on a 12-h light, dark cycle.

Zpl 2-1 and C6 cell lines were used for the neuronal cell marker and the glial cell marker, respectively. The neuronal cell line Zpl 2-1 was established from hippocampus of Zürich I mice, as previously described 16. The glial cell line, C6, was cloned from a rat glial tumor (ATCC CCL-107). Both cell lines were maintained in DMEM supplemented with 10% FBS, 100 unit/ml penicillin and 100 μg/ml streptomycin (Gibco BRL), incubated at 37°C in 5% CO₂.

Primary astrocyte cells were cultured from 1 day neonates from SAMR1, SAMP8, and ICR mice 17. Cells were obtained from neonates in full compliance with the ethical guidelines of the National Institutes of Health (NIH). Cells were cultured on 5 μg/ml poly-L-lysine (P-L-L; Sigma)-coated dishes with culture media (DMEM with 10% FBS, 100 unit/ml penicillin and 100 μg/ml streptomycin, Gibco BRL), incubated at 37°C in 5% CO₂ and transfected with SV40 large T antigen containing vector (φSV40; provided by Dr T. Onodera, Tokyo University) using 8 μg/ml of hexadimethrine bromide (Sigma-Aldrich, San Diego, CA, USA)18. After 24 h, cells were detached from culture dishes to eliminate microglia cells and oligodendrocytes and then transferred to new culture dishes. The origin of the mouse lines and characteristics of cell lines used in the present study are shown in Table 1.

For Western blot analysis, 50 μg protein from brain homogenates from each cell lysate and 40 μl of cell-free cell culture medium obtained after centrifugation at 25000 × g, 4°C for 30 min were separated on 12% Tris-glycine gels and transferred to nitrocellulose membrane (Amersham) 19. The membrane was blocked with 5% nonfat dry milk in 0.1% TBST (Tris-buffered saline with tween-20; 20 mM Tris-HCl, 140 mM NaCl, 0.1% Tween-20) for 1 h at room temperature and then probed with one of the following primary antibodies: rat-anti-GFAP (glial fibrillary acidic protein) at dilution of 1:5000 (DAKO, Glostrup, Denmark), mouse-anti-NeuN (neuron-specific nuclear protein) at 1: 1000 (Chemicon, Temecula, California, USA), mouse-anti-CD11b (Integrin α M) at 1:1000 (Serotec, Oxford, UK), mouse-anti-CNPase (2',3'-cyclic nucleotide 3'-phosphodiesterase) at 1:1000 (Sigma-Aldrich, St. Louis, Missouri, USA), and goat-anti-MuLV CAgag at 1:5000 (Quality Biotech, Inc.)1116. The primary antibody was incubated overnight at 4°C, and the appropriate secondary antibodies conjugated with horseradish peroxidase (Zymed, San Francisco, California, USA), anti-rat-HRP-conjugated at 1:3000, anti-mouse-HRP-conjugated at 1:5000, anti-goat-HRP-conjugated at 1:3000, were then added. Bound antibodies were visualized by chemiluminescence (Pierce, Rockford, Illinois, USA). Mouse-anti-β-actin at 1:10000 (Sigma-Aldrich, St. Louis, Missouri, USA) was used as a cellular marker. Expression levels of each protein were quantified by densitometer (GS-800, Bio-Rad, California, USA).

For immunocytochemistry, cells were plated on glass-cover slips and cultured for 24 h. Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) at room temperature for 10 min, treated with 5% normal donkey serum (Jackson, West Grove, Pennsylvania, USA) in PBS at room temperature for 1 h, and then rinsed with PBS. Cells were incubated with primary antibodies against rabbit-anti-GFAP at 1:100 (DAKO) and rat-anti-GFAP at 1:100 as an astrocyte marker, mouse-anti-MAP2 (microtubule-associated protein 2) at 1:50 (Upstate, Charlottesville, Virginia, USA) as a neuronal marker, mouse-anti-CD11b at 1:50 (Serotec) as a microglia marker and mouse-anti-CNPase at 1:50 (Sigma-Aldrich) as an oligodendrocyte marker, then maintained overnight at 4°C. Appropriate secondary antibodies conjugated with fluorochromes (Zymed), anti-rabbit-FITC at 1:200, anti-rat-FITC at 1:200, anti-goat-TRITC at 1:200 and anti-mouse-TRITC at 1:200, were then applied. After washing with PBS, cells were incubated with 10 μM DAPI (4',6-Diamidino-2-phenyindole, dilactate)(Sigma-Aldrich) at 37°C for 1 min and observed using confocal microscopy (Zeiss). DAPI staining was used as a cellular marker. For double-staining, cells on cover slips were prepared as noted above and then incubated with each primary antibody at the dilution listed above overnight at 4°C. After incubation, slides were washed with PBS and then appropriate secondary antibodies conjugated with fluorochromes (Zymed) were applied at the appropriate dilution as noted above. After washing with PBS, 10 μM DAPI staining was applied and the cells observed using confocal microscopy (Zeiss). The results were representative of at least three separate experiments.

Total mRNA was extracted using Trizol reagent (Invitrogen, Carlsbad, California, USA) and cDNA was synthesized from 2 μg of total RNA by reverse transcription using AMV reverse transcriptase (Promega, Madison WI) and oligo (dT) primer. To test for integration of SV40 large T antigen, genomic DNA was extracted from cultured cells using a DNA extraction kit (Qiagen, Hilden, Germany). PCR was performed with the following primers (Bioneer, Daejon, Korea): SV40 large T antigen, sense: 5'-TGAGGCTACTGCTGACTCT-3'; antisense: 5'-GCATGACTCAAAAAACTTAGCAATTCTG-3'; Akv, sense: 5'-ATGGAGAGTACAACGCT CTCA-3'; antisense: 5'-GAGGTTAGATTGTTGCTTACTG-3'. As a cellular marker GAPDH (glyceraldehydes 3-phosphate dehydrogenase) was performed with the following primers, sense: 5'-TGGTATCGTGGAAGGACTCATGAC-3'; antisense: 5'-ATGCCAGTGAGCTTCCC GTTCAGC-3'. Expression levels of Akv and GAPDH were quantified by densitometer (GS-800, Bio-Rad, California, USA). Purified RNA (2 μg) was used as a substrate for single-stranded cDNA synthesis. An aliquot (5 μl) of the cDNA of each sample was used for PCR with primers for IFNγ, TNF-α, TNF-β, IL-1α, IL-1β, IL-6, and β-actin, a housekeeping gene. The PCR primers used are shown in Table 2202122. The DNA mixture was amplified for 30 cycles (each consisting of denaturation 45 sec at 95°C, annealing for 45 sec at 58°C, extension for 1 min at 72°C), received a final extension for 10 min at 72°C and was stored at 4°C in a thermal cycler (Amplified Biosystem, USA) 2122. Products were analyzed by 1% agarose gel electrophoresis and visualized by ethidium bromide staining under UV light.

SC-1 cells (ATCC CRL-1404) were grown in Dulbecco's modified eagle medium (DMEM) + 10% fetal bovine serum (FBS) + 100 unit/mL penicillin + 100 μg/mL streptomycin (DMEM10A). The XC cell line (ATCC CCL-165) was grown in DMEM + 10% FBS without antibiotics (DMEM10). SC-1 and XC cells were harvested for use in plaque assays using trypsin and suspended in the appropriate medium for assay. All cell growth and plaque assays were done at 37°C in a 5% CO₂ incubator.

Cells were harvested by trypsinization and kept on ice until further processing by homogenization in DMEM (10% w/v), using 20 strokes in a hand-operated tissue homogenizer. Serial dilutions of cell homogenates were prepared in DMEM + 5% FBS + penicillin-streptomycin + 25 μg/mL DEAE-dextran (DMEM5A-DEAE).

Ecotropic MuLV was quantitated using the SC-1/UV plaque assay 10. SC-1 cells were plated onto 60 mm dishes at 10⁵ cells/dish in 4 mL DMEM10A. The next day, 1 h before the addition of cell homogenates, medium in the dishes was discarded and replaced with 3 mL DMEM5A-DEAE. One mL of sample (brain homogenate or cell line homogenate) diluted in the same medium was then added to plates. One to 2 days after addition of homogenates, medium was removed and replaced with 4 mL/dish DMEM5A. After 5 days, medium was removed and cultures were exposed to 30 s of UV irradiation. Immediately after UV irradiation, 4 mL of a suspension containing 3.0 × 10⁵ XC cells/mL in DMEM10 were pipetted into each dish. After 24 h incubation, the medium was discarded. Cultures were washed once with phosphate-buffered saline (PBS), fixed with 100% methanol for 5 min and stained with hematoxylin (Fisher Scientific, USA) for 5 min. Hematoxylin was discarded, the cultures washed twice with tap water, and the plaques counted under a dissecting microscope.

Cells were fixed in half-Karnovsky's fixative (1.66% glutaraldehyde, 1.6% paraformaldehyde buffered with 0.1 M cacodylate buffer) for 2 h at 4°C. Post-fixation was done in 1% osmium tetroxide buffered with cacodylate buffer for 1.5 h at 4°C. Following fixation, cells were pelleted and washed three times in 0.1 M cacodylate buffer. Cell pellets were dehydrated through a graded ethanol-propylene oxide series and embedded in Epon 812 (Electronic Microscopy Science). Ultra thin sections (75 nm) were cut in a RMC MTXL ultramicrotome (Tucson, USA) and stained with 2% uranyl acetate and lead citrate. The sections were observed using a transmission electron microscope (Zeiss-EM109, Oberkochen, Germany).

For immuno-electron microscopy experiments, cells were fixed for 3 h at 4°C with 3% paraformaldehyde and 0.25% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4). Pellets were dehydrated in increasing concentrations of ethanol and embedded in LR white (Electron Microscopy Sciences, Hatfield, PA, USA) and cured for 48 h at 50°C. Ultra thin sections (75 nm) were collected on nickel grids and used for labeling with a goat antiserum specific for the virus CAgag protein, followed by incubation with a rabbit anti-goat IgG conjugated to 15 nm gold particles (Electron Microscopy Sciences, Hatfield, PA, USA). Sections were post-stained with 2% uranyl acetate and lead citrate and observed with a transmission electron microscope (Zeiss-EM109, Oberkochen, Germany). The viral particles were counted and their diameters measured in 13 independent fields (85 μm²) of R1A and P8A cell lines. The number of gold particles were counted in 18 independent fields (85 μm²) within the plasma membrane.

To obtain negative staining images, cells were scraped then frozen and thawed for three cycles. Supernatant and disrupted cells were combined and centrifuged at 1000 × g for 10 min at 4°C. Supernatants were transferred to a 20% sucrose cushioned tube and centrifuged at 130000 × g for 3 h at 4°C (SW28 rotor, Beckman). Viral particles were suspended in 30 μl of sterile phosphate-buffered saline (PBS) then incubated at 37°C for 30 min. Fixation and staining were done using 2% phosphotungustic acid (PTA) solution.

Each of the cell lines was seeded at a density of 1 × 10⁵ cells and incubated at 37°C in 5% CO₂; a series of separate cell cultures were stained with 0.4% trypan blue solution (Sigma-Aldrich, USA) and counted each day for 12 days using hemacytometer (Sigma-Aldrich, USA)(Kim et al., 2005). Cell growth and morphological analysis was assessed using inverted microscopy (Zeiss, Oberkochen, Germany). Each cell count and microscopic analysis was repeated at least three times.

Statistical analyses were performed by appropriate one-way ANOVA test. All data were reported as means ± SD. A P value of less than 0.05 was considered significant.

The expression levels of the MuLV gene and protein were investigated in brains of 12 months ICR, SAMR1, and SAMP8 mice. Using RT-PCR analysis of endogenous MuLV gene expression, we have shown that the expression of MuLV was not detected in ICR nor SAMR1 brains (R1B) but was present in SAMP8 brains (P8B) (Fig. 1A). MuLV gene expression level in P8B was significantly higher than ICR and R1B (p < 0.01) (Fig. 1B). To analyze the expression level of the MuLV protein, CAgag, a Western blot was performed. In agreement with RT-PCR data, MuLV protein was neither detected in ICR nor R1B but was present in P8B (p < 0.01) (Fig. 1C and 1D). GAPDH and β-actin were included as concentration monitors for RT-PCR analysis and Western blot, respectively.

Three distinct astrocyte cell lines were established from the cerebral region of SAMR1 (R1A cell lines: R1A1, R1A2 and R1A5), SAMP8 (P8A cell lines: P8A1, P8A7 and P8A9), and ICR (ICR-A cell lines: ICR-A1, ICR-A2 and ICR-A3) mice (Table 1). The presence of the gene for SV40 large T antigen was examined with PCR analysis using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the housekeeping control gene. As shown in Fig. 2A, established R1A, P8A, and ICR-A cell lines had similar expression levels of SV40 large T antigen as an immortalization marker. The cell-type marker antibodies described in Materials and Methods were used to characterize the transformed cell lines. SAMP8 mouse brain was positive for both the astroglial (GFAP) and the neuronal (NeuN) marker. Zpl 2-1 and C6 cell lines were used as positive controls for the neuronal marker and the astroglial marker, respectively. R1A, P8A, and ICR-A cell lines were astroglial-positive in Western blot analysis using antibodies against GFAP, a 50 kDa protein band which did not react with Zpl 2-1 hippocampal neuronal cell lysates (Fig. 2B). Using anti-NeuN antibody, a 66 kDa protein band was detected only in Zpl 2-1 cell lysates and SAMP8 brain homogenates (Fig. 2B). Thus, the established cell lines were shown to be composed of astroglial cells. The results of immunofluorescence analysis were in accordance with the Western blot findings: R1A, P8A, ICR-A, and C6 cell lines were positive for GFAP staining but not MAP-2 staining, whereas Zpl 2-1 cells were positive for MAP-2 but not GFAP (Fig. 3). All cultures were negative for the oligodendrocyte and microglial markers in both Western blot and immunofluorescence experiments (data not shown).

RT-PCR analysis was used to assess the level of endogenous MuLV expression in R1A, P8A and ICR-A cell lines. Homogenate of SAMP8 brain was used as a positive control and Zpl 2-1 cell line and SAMR1 brain homogenate served as negative controls. We found that MuLV expression was not detected in ICR-A or Zpl 2-1 cell lines, nor in SAMR1 brain (R1B) but was present in R1A and P8A cell lines and SAMP8 brain (P8B) tissue (Fig. 4A). The expression level of MuLV in P8A cell lines was significantly higher than in R1A cell lines (p < 0.01) (Fig. 4B). In contrast, GAPDH was expressed to the same extent in all samples.

In order to analyze the expression of the MuLV protein, CAgag, Western blot analysis was performed. CAgag was detected in SAMP8 brains, R1A cell lines, P8A cell lines, and in SC-1 cells which had been infected with P8A cell line homogenate (labeled SC-1-Tf-P8A1), whereas CAgag was not detected in the Zpl 2-1 cell lines, SAMR1 brains, and ICR-A cell lines (Fig. 5A). The CAgag levels in P8B cell lines were significantly higher (p < 0.01) than in R1A cell lines (Fig. 5B). SAMR1 brain homogenate and Zpl 2-1 cell homogenate served as negative controls and SAMP8 brain homogenate was used as a positive control. The same amount of total protein was analyzed as shown by the similar levels of β-actin expression in all of the samples. In agreement with the Western blot findings, the R1A and P8A cell lines were positive for CAgag immunostaining, whereas ICR-A cell lines were negative (Fig. 5C). Staining for endogenous MuLV protein was detected in cytoplasm where it was merged with GFAP staining. In accordance with Western blot analysis, the amount of CAgag staining was less in R1A cell lines than in P8A cell lines. The difference in color of the merge pictures seen for the R1A1 and P8A9 is probably a function of the fact that the concentration of CAgag is higher in the P8 cell lines than in the R1 cell lines.

To determine whether cell-associated CAgag detected in homogenates of R1A and P8A cells was released from cells, culture media of each cell line was harvested and analyzed using anti-CAgag antibody in Western blots. CAgag was detected in R1A cell lines at low levels and in P8A cell lines at high levels (Fig. 6A). There was a significant difference in the level of released CAgag between the two types of cell lines (p < 0.01) (Fig. 6B). The level of CAgag found in P8A9 cell lysate (P8A9-Cl in Fig. 6B) was slightly less than that seen in the media from P8A9 cell culture. Zpl 2-1 culture media was used as a negative control and SAMP8 brain homogenate was the positive control.

Using transmission electron microscopy (TEM), synthesized virus particles budding from the plasma membrane were observed in R1A and P8A cell lines (Fig. 7A and 7B); similar particles were not observed in ICR-A cell lines (data not shown). The concentration of MuLV particles was significantly higher in P8A cell lines than in R1A cell lines (p < 0.01), as determined by either uranyl acetate/lead citrate staining or by gold-labeling (Fig. 7C). The diameters of particles in the fixed tissues were measured in 13 independent fields (85 μm²), and each experiment was repeated three times. Viral particles observed in R1A cell lines averaged 70 nm including their envelope, whereas particles from P8A cell lines averaged 80 nm.

For PTA-stained virus preparations derived from the different cultures, the different morphology seen in virus particles from R1A and P8A shown in Fig. 7D is not a consistent difference between the two cell cultures, but rather reflects the variation seen in PTA preparations: the R1A particle morphology shown in Fig. 7D can also be seen in P8A preparations and vice-versa.

Testing of R1A, P8A, and ICR-A cell lines for ecotropic MuLV infectivity showed striking differences in virus titer among these cell lines (Table 3). R1A and ICR-A cell lines exhibited no demonstrable virus in either cells or supernatants even using undiluted inoculum, whereas 10⁴~10⁶ plaque-forming units (PFU)/ml of virus was found in P8A cells and supernatants.

The cell morphology of the 3 types of cells is different: The R1A cells have a long hexagonal shape with smooth plasma membranes, P8A cells have a pentagonal shape with ruffled plasma membranes, and ICR-A cells have a pentagonal shape with smooth plasma membranes (Fig. 8).

R1A, P8A, and ICR-A cell lines also showed differences in their growth rates. For the first 7 days, the proliferation rates for the R1A cell lines were higher than those for the P8A and ICR-A cell lines (Fig. 9A); this was reflected in an analysis of the doubling times for the cell lines (Fig. 9B). The growth rates for P8A and ICR-A cell lines were virtually identical prior to D7 and, therefore, their doubling rates were similar. After D7, cell numbers for R1A and ICR-A cell lines remained relatively constant, whereas the P8A cell lines showed a dramatic increase in cell counts until D10 and then decreased on Days 11 and 12 (Fig. 9A).

The levels of gene expression of the following inducible cytokines were measured in the 3 cell types: IFN-γ, TNF-α/β, IL-1α/β, IL-6 and iNOS. The results of RT-PCR analysis showed marked induction of TNF-β, IL-β, and IL-6 in R1A cell lines, IFN-γ, TNF-α, and IL-1α in P8A cell lines, and IFN-γ, TNF-α and TNF-β in ICR-A cell lines (Fig. 10A). Expression levels were quantitated and compared by densitometry (Fig. 10B); iNOS was not activated in any astroglial cell lines used in this study (data not shown).

Significant differences in expression between R1A and P8A cell lines were seen in IFN-γ, TNF-α and TNF-β, IL-1α, IL-1β, and IL-6 (Fig. 10B). Significant differences between P8A and ICR lines were seen for TNF-α and IL-1α.