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. 2004 May;78(10):5088-96.
doi: 10.1128/jvi.78.10.5088-5096.2004.

Differential cell killing by lymphomagenic murine leukemia viruses occurs independently of p53 activation and mitochondrial damage

Suparna Nanua  1 , Fayth K Yoshimura
Affiliations

Differential cell killing by lymphomagenic murine leukemia viruses occurs independently of p53 activation and mitochondrial damage

Suparna Nanua et al. J Virol. 2004 May.

Abstract

Upon inoculation into AKR mice, mink cell focus-forming murine leukemia virus (MCF MLV) accelerates thymic lymphoma formation. During the preleukemic phase of disease, we observed the induction of apoptosis in thymic lymphocytes. A similar induction of apoptosis was observed for cultured mink epithelial cells after MCF13 MLV infection. In this study, the relevance of viral pathogenicity to cell killing was determined by testing the susceptibility of various cell types from different species to lymphomagenic MLVs. We observed that the cytopathic effect of lymphomagenic MLVs was restricted to mink cells. Southern blot analysis of MLV-infected cells revealed an accumulation of the linear form of unintegrated viral DNA, particularly in mink cells after MCF13 MLV infection. Thus, a strong correlation was observed between viral superinfection, which results in the accumulation of high levels of unintegrated viral DNA, and cell killing. Immunoblot analysis for MCF13 MLV-infected mink epithelial cells did not show a significant change in total p53 levels or its phosphorylated form at Ser-15 compared with that in mock-treated cells. Moreover, a time course analysis for mink epithelial cells infected with MCF13 MLV did not reveal mitochondrial depolarization or a significant change in Bax levels. These results demonstrate that MCF13 MLV induces apoptosis preferentially in cells in which superinfection occurs, and the mechanism involved is independent of p53 activation and mitochondrial damage.

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Figures

FIG. 1.
FIG. 1.
Effect of MLV infection on mink epithelial cell growth. (A) Mink epithelial cells were infected with either MCF247 MLV at an MOI of 1 (•) or 7 (▴), MCF13 MLV at an MOI of 7 (▪), or Mut-MCF13 MLV at an MOI of 7 (X). Growth of mock-infected cells is also shown (⧫). (B) Mink epithelial cells were infected with NZB-9 MLV at an MOI of 1 (•) or 7 (▴) or MCF13 MLV at an MOI of 7 (▪) or were mock infected with medium (⧫). Cells were trypsinized at the indicated days after infection, and viable cells were enumerated by trypan blue dye exclusion. Values represent the means and standard deviations calculated from counting duplicate samples from two independent experiments.
FIG. 2.
FIG. 2.
High levels of linear UVD in mink cells compared with levels in M. dunni and NIH 3T3 fibroblasts after MCF13 MLV infection. (A) Southern blot analysis of Hirt-extracted DNA from MCF13 MLV-infected mink epithelial cells (lanes 5 to 8), mink fibroblasts (lanes 9 to 11), and M. dunni fibroblasts (lanes 12 to 15) at an MOI of 0.5 and from NIH 3T3 fibroblasts (lanes 16 to 18) at an MOI of 7 at the indicated hours postinfection (hr p.i.). Plasmid DNA corresponding to 160, 40, and 10 pg of genomic-length MCF13 MLV was electrophoresed in lanes 2, 3, and 4, respectively. Hybridization was done with a 32P-labeled 8.2-kb length of MCF13 MLV genomic DNA. Lane 1 contains the DNA ladder. (B) Copy number per cell of the linear form of UVD over time for MCF13 MLV-infected mink epithelial cells (⧫), mink fibroblasts (▪), M. dunni fibroblasts (▴), and NIH 3T3 fibroblasts (X). Data are representative of at least two independent experiments.
FIG. 3.
FIG. 3.
Low levels of linear UVD in mink epithelial cells infected with NZB-9 MLV. (A) Southern blot analysis of Hirt-extracted DNA from mink epithelial cells infected with NZB-9 MLV at an MOI of 1.2 (lanes 5 to 8) or MCF13 MLV at an MOI of 0.5 (lanes 9 to 12) was performed at the indicated hours postinfection (hr p.i.). Lanes 2, 3, and 4 consist of 160, 40, and 10 pg, respectively, of plasmid DNA corresponding to genomic-length MCF13 MLV. Lane 1 contains a DNA ladder. (B) Copy number per mink epithelial cell of the linear form of UVD for either MCF13 MLV at an MOI of 0.5 (⧫) or NZB-9 MLV at an MOI of 1.2 (▪) at the indicated times after virus infection. Data are representative of two independent experiments.
FIG. 4.
FIG. 4.
MCF13 MLV infection does not result in p53 activation. (A) Western blot analysis for total p53 levels in cellular extracts from mink epithelial cells either mock treated (M; lanes 1, 3, 5, 7, 9, and 11) or infected with MCF13 MLV at an MOI of 5 (V; lanes 2, 4, 6, 8, 10, and 12) at the indicated times postinfection (hr p.i). Total cellular extracts from mink epithelial cells exposed to γ-irradiation (γ-IR; lane 14) or untreated cells (C; lane 13) were used as controls. (B) Western blot analysis for phosphorylated p53 (Ser-15) on cellular extracts from mink epithelial cells either mock treated (M; lanes 2, 4, 6, 9, 11, and 13) or infected with MCF13 MLV at an MOI of 5 (V; lanes 3, 5, 7, 10, 12, and 14) at the indicated times postinfection (hr p.i.). A phospho-p53 (Ser-15)-specific antibody was used. Total cellular extracts obtained from mink epithelial cells exposed to either 20 mM hydroxyurea (HU; lanes 1, 8, and 16) or γ-irradiation (γ-IR; lane 18) or from untreated cells (C; lanes 15 and 17) were used as controls.
FIG. 5.
FIG. 5.
Absence of mitochondrial depolarization in mink epithelial cells infected with MCF13 MLV. Mink epithelial cells were either mock treated (M; a, c, e, and g) or infected with MCF13 MLV as described for Fig. 6 (V; b, d, f, and h). At the indicated time points postinfection, cells were stained with 200 nM MitoTracker Red and imaged with a Zeiss Axiophot fluorescence microscope. Mink epithelial cells treated with 1 μg of valinomycin per ml for 1 h (Val; j) or untreated (C; i) were used as controls.
FIG. 6.
FIG. 6.
No change in Bax levels was observed after MCF13 MLV infection. Results shown are from a Western blot analysis of Bax in total cellular extracts from mink epithelial cells that were either mock treated (M; lanes 2, 4, 6, 8, 10, and 12) or infected with MCF13 MLV at an MOI of 5 (V; lanes 3, 5, 7, 9, 11, and 13) at the indicated times postinfection (hr p.i.). Total cellular extracts from mink epithelial cells exposed to 20 mM hydroxyurea (HU; lanes 1 and 15) or untreated (C; lane 14) were used as controls.
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