Activity of Ras and its downstream signaling pathways in mouse MPNST cells. A: Schematic representation of Ras and its downstream effector pathways. B: The activity of Ras in different mouse MPNST cell lines was detected by Ras affinity pull-down assay for Ras-GTP. The first panel shows the amount of activated form of Ras (Ras-GTP), and the second panel shows the amount of total form of Ras. Western blotting using phospho-specific antibody against ERK1/2 was used to detect the amount of activated ERK in MPNST cells in comparison to the amount of total form of this enzyme. The activities of other Ras downstream effector pathways were investigated by pull-down assays coupled with in vitro kinase reactions for JUN, P38, and PI3K pathways. In each assay an in vitro kinase reaction was performed to detect the capability of each effector in phosphorylating its specific substrate, which was in turn detected by blotting with a phospho-specific antibody. Cell lines are numbered as follows: lane 1, 6IE4; lane 2, 37-3-18; lane 3, 35-1-2; lane 4, 38-2-18; lane 5, 32-5-24-12.

Permissiveness of MPNST cells to infection by HSV-1(F) and G207. A: The released progeny virus was titrated after exposure of different MPNST cell lines as well as Vero cells to G207 (an oncolytic version of HSV-1, white columns) or the parental virus, HSV-1, strain F (black columns), at the 20th hour after infection at MOI ∼ 2. The number of plaques/ml (as a representation of infectious viral particles) was counted after staining with Giemsa in independent experiments. B: Morphology of MPNST cells lines in the presence and absence of G207 (MOI ∼ 2) virus for 24 hours as studied by light microscopy (arrows point to monolayer showing rounding and clumping typical of herpetic infection). C: Expression of HSV-1 glycoprotein-C (gC) in G207-infected MPNST cells. A FITC-conjugated mouse monoclonal antibody was used to detect the expression of the HSV-1 glycoprotein C (gC) in different MPNST cell lines 24 hours after infection with G207. The green color represents expression of this viral envelope protein. D: Western blot analysis of HSV-1 proteins produced in G207-infected MPNST cells. The viral protein production was evaluated in five MPNST cells after 24 hours of infection with G207 using a polyclonal antibody raised against HSV-1 antigens. Control 6IE4 cells (lane 6) were not infected and served as an indicator for the specificity of the antibody. E: Proliferation of 37-3-18 and 38-2-18 cells after infection with G207 (MOI ∼ 1) was compared to uninfected control cells at 48 hours after infection (control cells are plotted as 100%). Callout panels show the density of cells at this time point. F: Invasiveness of 37-3-18 cells and 38-2-18 cells after infection with G207 (MOI ∼ 1) was compared to uninfected control cells at 48 hours after infection (control cells are plotted as 100%). Notably, control 38-2-18 cells were much less invasive than control 37-3-18 cells. Callout panels show the density of invaded cells at this time point. G: Effects of Ras signaling inhibitors on production of G207 proteins in the 37-3-18 cell line. One of the most permissive MPNST cell lines, 37-3-18, was exposed to different inhibitors of Ras signal transduction pathway overnight and then infected with the G207. Cells were lysed after 24 hours after infection and the amount of viral proteins in the lysates was detected by Western blotting. Inhibitors were used at the following concentrations: AG1478 (an EGFR blocker) at 0.5 μmol/L, L-744,832 (a Ras farnesylation blocker) at 60 μmol/L, PD98059 (an ERK pathway blocker) at 20 μmol/L, and SP600125 (a JNK-pathway blocker) at 25 μmol/L. Cell lines are numbered as follows: lane 1, 6IE4; lane 2, 37-3-18; lane 3, 35-1-2; lane 4, 38-2-18; lane 5, 32-5-24-12. Scale bars: 600 μm (B, E, F); 300 μm (C).

Permissiveness of Schwann cells to HSV-1(F) and G207. A: SW10 mouse Schwann cells were incubated at 33°C (proliferative because of SV40-LT expression) and 37°C (nonproliferative) and infected with HSV-1(F) and G207 (MOI ∼ 2). At 24 hours after infection with HSV-1(F), cells were studied with light microscopy for appearance of morphology of infection (middle) as well as stained with a FITC-conjugated mouse monoclonal antibody against expression of the HSV-1 glycoprotein C (gC, green spots) as a marker for herpetic infection (right). Morphological changes after infection can be compared with uninfected cells shown in the left panels. B: SW10 cells cultured at different temperatures are infected with G207 virus and the morphological changes (left) as well as expression of gC (right, green spots) were detected by microscopy. The red background staining in bottom right panel is because of the Texas Red component of the antibody preparation that stains all cells. This panel is photographed at Texas Red channel to visualize the monolayer confluency. C: The amount of viral protein synthesis in SW10 cells for each virus in different temperature conditions (proliferative because of SV40-LT expression at 33°C and nonproliferative at 37°C) was evaluated by Western blotting for herpes proteins. D: Using affinity pull-down assays, the amount of Ras-GTP (active form of Ras) in SW10 cells at proliferative (33°C) or nonproliferative (37°C) temperatures was evaluated. E: The effects of treatment with a specific Raf-kinase inhibitor (500 nmol/L) on viral protein synthesis in SW10 cells infected with HSV-1(F) and G207 at 33°C was studied by Western blotting for herpes proteins. Scale bars = 600 μm.

Induction of apoptosis after UV exposure in MPNST cells. MPNST cells with high- and low-Ras signaling activity were exposed to UV (10 mJ/cm²) and the induction of apoptosis was measured after 6 hours using the Annexin-V conversion test. A: The average (±SD) percentage of apoptosis occurring after UV exposure is plotted for two groups including high-Ras (6IE4 and 37-3-18) and low-Ras MPNST cells (35-1-2, 38-2-18, and 35-5-24-12). B: Annexin-V labeling results obtained for 37-3-18 and 38-2-18 cells after exposure to UV. The top right quadrant represents cells committed to undergoing apoptosis.

Induction of apoptosis after infection of MPNST cells with G207. A: Nuclear morphology of infected cells was studied by 4′,6-diamidino-2-phenylindole (DAPI) staining (the red background in bottom right panel is because of the Texas Red component of anti-gC antibody preparation) (arrows show nuclei with chromatin condensation). B: Different MPNST cell lines were infected with G207 (MOI ∼ 2). Twenty-four hours after infection the occurrence of apoptosis was evaluated using TUNEL assay. The cells were fixed on a slide and studied with a fluorescence microscope for FITC labeling of nucleosome-sized DNA. C: The panels show fluorescence-activated cell sorting data as the counts of sorted cells (fluorescent events) versus the intensity of FITC used for labeling the nucleosome-sized DNA in MPNST cells undergoing apoptosis after 24 hour of infection with G207. D: The graph shows the percentage (mean ± SD) of apoptotic cells for each MPNST cell line after 24 hour of exposure to G207. Cell lines are numbered as follows: lane 1, 6IE4; lane 2, 37-3-18; lane 3, 35-1-2; lane 4, 38-2-18; lane 5, 32-5-24-12. Scale bars = 300 μm.

Ras signaling pathway influences the outcome of oncolytic virus G207 insult in MPNST cells. This diagram explains the influence of Ras signaling via its downstream effector pathways on permissiveness of MPNST cells to G207. In cells with low levels of Ras signaling, viral protein synthesis does not occur at an efficient level. However, exposure to virus does induce certain levels of apoptosis in these cells in part because of an inactive PI3K pathway failing to exert its anti-apoptotic effects. In cells with elevated levels of Ras signaling, however, efficient viral protein synthesis and activation of the PI3K pathway reduces the capability of cells to undergo apoptosis. The expected outcome of this scenario would be robust G207 replication and death of cells via lytic infection (necrosis).