Astrocytes Survive Chronic Infection and Cytopathic Effects of the ts1 Mutant of the Retrovirus Moloney Murine Leukemia Virus by Upregulation of Antioxidant Defenses

Bcl-2 in C1 versus C1-ts1 cells and in C1 versus C1-ts1-S cells. C1, C1-ts1, and C1-ts1-S cell lysates were prepared after 24, 48, and 72 h of incubation or after infection. Bcl-2 and catalase levels were analyzed by Western blotting. The blots were then stripped and reimmunoblotted with anti-β-actin antibody. (A) Bcl-2 levels in uninfected C1 cells versus C1-ts1 cells assayed at 24, 48, and 72 hours p.i. (B) Bcl-2 and catalase levels in uninfected C1 cells versus C1-ts1-S cells at 24, 48, and 72 h of culture. The results are representative of three separate experiments. “Fold” values appearing under the bands are ratios of band density, measured by densitometric tracing, to the band density for C1 control cells after normalization for protein loading using the β-actin bands.

gPr80^env and gp70, GRP78, GRP94, and CalR protein expression in C1 and C1-ts1-S cells. C1 or C1-ts1-S cells were plated as described in Materials and Methods, but in separate sets of replicate cultures, with one set left untreated and the other treated with 1 mM BSO for the last 24 h of incubation. After incubation for 24, 48, and 72 h, cell lysates were prepared, and 50 μg of total protein from each lysate was analyzed by Western blotting for gPr80^env and gp70, GRP78, GRP94, and CalR. The blots were then stripped and reimmunoblotted with anti-β-actin antibody. The results are representative of two or three experiments. The values under the bands are ratios of band density, measured by densitometric tracing, to the band densities for C1 control cells after normalization of protein loading using the β-actin bands.

Intracellular H₂O₂ in C1 and C1-ts1-S cells. Intracellular H₂O₂ levels were measured via CM-H₂DCFDA fluorescence in uninfected and acutely infected C1 cells or C1-ts1-S cells. After incubation for 24, 48, and 72 h, all cells were loaded with 10 μM CM-H₂DCFDA for 30 min, followed by flow cytometric measurement of their H₂O₂ contents. The three panels represent the results for cells harvested at 24 (top), 48 (middle), and 72 (bottom) hours p.i. Shown are uninfected C1 cells (C1; blue lines), acutely ts1-infected C1 cells (ts1; green lines), C1-ts1-S cells (C1-ts1-S; red lines), and BSO-treated C1 cells (BSO; brown lines).

Intracellular GSH and cystine/cysteine levels, cystine uptake, and γ-GT activities in C1 and C1-ts1-S cells. (A) GSH levels in C1 cells, C1-ts1-S cells, and acutely infected C1 cells. GSH levels were determined for each culture using a GSH assay kit following the manufacturer's instructions. *, P < 0.05; **, P < 0.01, comparing C1-ts1-S cells with C1 cells. #, P < 0.05; ##, P < 0.01, for acutely infected C1-ts1 cells versus C1-ts1-S cells. (B) Intracellular cystine/cysteine levels in C1 and C1-ts1-S cells. Total intracellular cysteine was measured by fluorometric HPLC. *, P < 0.05; **, P < 0.01; ***, P < 0.001, comparing C1-ts1-S and C1-ts1 cells with C1 cells at 24, 48, and 72 h, respectively. #, P < 0.05; ###, P < 0.001, for C1-ts1 cells versus C1-ts1-S cells. (C) Uptake of [³⁵S]cystine at 24 hours p.i. Cells were incubated in bicarbonate buffer for 20 min prior to the addition of 0.5 μCi/ml of L-[³⁵S]cystine. The cells were then incubated for 3 min, washed, and solubilized for liquid scintillation counting and for protein determination. [³⁵S]cystine uptake rates are shown. **, P < 0.01, for C1-ts1-S cells versus C1 cells. (D) γ-GT activities in C1 and C1-ts1-S cells at 24 hours p.i. C1 or C1-ts1-S cells were either left untreated or treated with 200 μM of acivicin for the last 4 h of their 24-hour incubation. γ-GT enzymatic activity in whole-cell lysates was determined using a commercially available γ-glutamyl transferase kit, according to the manufacturer's instructions. γ-GT activity was taken from a standard curve. ***, P < 0.001, for untreated C1-ts1-S cells versus untreated C1 cell controls. ###, P < 0.001, for acivicin-treated C1 cells versus untreated C1 cell controls. +++, P < 0.001, for acivicin-treated C1-ts1-S cells versus untreated C1-ts1-S cell controls. All bars reflect means plus or minus standard deviations from two to four independent experiments.

Transient upregulation of Nrf2 and activation of its downstream target genes xCT, γ-GCL subunits, and GPx in C1-ts1-S cells. (A) C1 or C1-ts1-S cells were incubated for 24, 48, and 72 h in the presence or absence of BSO for the last 24 h of their incubation. For cells harvested at each time point, nuclear lysates were prepared and analyzed for their content of Nrf2. (B) C1 or C1-ts1-S cells were incubated for 24, 48, and 72 h in the presence or absence of BSO for the last 24 h of their incubation. For cells harvested at each time point, nuclear lysates were prepared and analyzed for their content of xCT, γ-GCLC and γ-GCLM, and GPx. All blots were stripped and reimmunoblotted with anti-β-actin antibody as a protein loading control. All blots shown are representative of three or four independent experiments. The values under the bands are ratios of band density, measured by densitometric tracing, to the band densities of C1 control cells after normalization of protein loading using the β-actin bands.

Cell numbers and GSH levels at 24, 48, and 72 h of culture in C1 and C1-ts1-S cells grown in cystine-deficient medium. C1 or C1-ts1-S cells were plated in 60-mm dishes and cultured in cystine-free DMEM with 10% FBS, supplemented with the indicated concentrations of cystine, and incubated for 24, 48, and 72 h. (A) Cell viability assays at 24 (top), 48 (middle), and 72 (bottom) hours p.i. Viable cells were counted by trypan blue exclusion. The results are the means plus standard deviations for three different experiments. (B) Intracellular GSH levels, measured as described in Materials and Methods, for cells grown as described for panel A, for 24 (top), 48 (middle), and 72 (bottom) hours p.i. The results are the means plus standard deviations for three different experiments.