The effects of SS1 infection on the phenotype of prostate cells.

<p>The proliferation rate of prostate cells is decreased upon exposure to SS1 virus (MOI∼1) with lower levels observed in case of prostate cancer cells with high Ras signaling (LnCap and LapC4) and PrEC and BPH-1 cells as compared to Du145 and PC3 cells (low Ras cells). (A) Invasiveness of prostate cancer cells LapC4 and Du145 as well as BPH-1 cells were decreased after 24 hours of exposure to SS1 virus in a significant manner. The decrease in invasiveness was more prominent for cells with elevated Ras signaling (LapC4 and BPH-1) as compared with Du145. Control Du145 cells were much less invasive than other cells. (B) Increase in necrosis and apoptosis is observed upon exposure of prostate cells to SS1 virus. The induction in necrosis/apoptosis is remarkably greater in LnCap and LapC4 cells. (C) The progression of cell cycle is altered upon exposure of prostate cancer cells to SS1 virus. In case of a high Ras cell (LapC4) an increase in G1 and decrease in S and G2 was observed upon infection with SS1 as compared with parental virus infected and control cells. Du145 (a low Ras cell), however, showed a passage through G1 but significant enhancement of S1 fraction. Left panels represent captured data plotted as fluorescence intensity (FL2-H channel) versus cell number for different phases of cell cycle. The right panel portrays these data as percentage of control (non-infected cells) for SS1 or HSV-1 infected cells. (D) Colony formation capability of LapC4 and Du145 cells was also significantly reduced upon infection with SS1. Upper panels show formation of colonies for a range of number of inoculated infected cells. The lower panels show average number of colonies per microscopy field for SS1-infected and control groups. The colony forming capability of Du145 cells were less inhibited as compared to LapC4 cells. (E) The mechanism of SS1 action is illustrated in this figure. Activation of Ras signaling pathway stimulates a signal through Raf/MEK/ERK pathway inducing phosphorylation of ELK. Stimulation of SRE elements by a complex including P-ELK results in expression of ICP4 and replication of SS1 which eventually destroys the cell via infection.</p

SS1 virus preferentially infects cells with increased Ras/ELK signaling.

<p>Morphological studies of SS1-infected cells (MOI∼1) show rounding and clumping (signs of herpetic infection) in cancer cells with elevated Ras/ELK signaling (LnCap and LapC4) as well as PrEC and BPH-1 cells as compared with Du145 and PC3 cells at 24-48 hours post-infection (MOI∼1). (A) The titration of viral progeny at 24–48 hours post-infection (MOI∼1) revealed enhanced levels for LnCap and LapC4 as well as PrEC and BPH-1 as compared with Du145 and PC3 cells. In case of PrEC and BPH-1 cells titrations maximize at 24 hours. (B) Immunofluorescent (IF) studies for glycoprotein C (gC, a marker for herpetic infection) revealed enhanced expression for LapC4 (high Ras) as compared to Du145 (low Ras) cells upon infection with SS1 (second row). Expression of this marker in these two cell lines was at much closer levels once these cells were infected with parental HSV-1. Top panel represents IF intensity for each panel versus number of captured events in each field. Third row represents the DAPI staining of related panels. Bottom panel shows staining of uninfected Du145 cells and the background Texas-red and DAPI staining. (C) The expression of SS1 proteins was investigated by western blotting on lysates from infected and control cells using an anti-body raised against all HSV-1 antigens. Higher and more comprehensive levels of SS1 protein synthesis was observed for LnCap, LapC4, PrEC and BPH-1 as compared to Du145 and PC3 cells. Controls (uninfected cells) show no bands proving the specificity of antibody for viral proteins. Lower panel shows a significant decrease in the expression of SS1 proteins in infected LnCap cells upon exposure to inhibitors of Ras (FTI-277 at 20 µM), MEK (PD98059 at 25 µM) and ELK (Chromomycin A at 10 µM) but not the vehicle (DMSO). A lower level of inhibition was observed for inhibitor of EGFR, AG1478 (0.5 µM). Cells were incubated with these inhibitors overnight and then exposed to SS1 while inhibitors existed in the media.</p

Characteristics of Signal-Smart 1(SS1) mutant HSV-1.

<p>The SS1 virus contains one copy of ICP4 gene under the control of 5xSRE and minimal TATA sequence. (A) Viral genomic PCR reactions confirm the structure of the recombinant ICP4 gene. The sequence for each pair of primer, the relative positions and of the primers as well as their sequence and results are shown. The PCRs were performed on U87 cells (non-transfected), d120-genomic DNA (labeled as d120) and the plasmid pTSIIDT. Performing PCR on back-bone elements of pTSIIDT is done in order to rule out the contamination of genomic viral DNA preparations by pTSIIDT. (B) Genomic structure of HSV-1 and SS1 virus and pTSIIDT plasmid DNA. NotI restriction analysis: The 9.3 Kb NotI fragment contains the plasmid backbone, the 2.8 and 0.7 Kb fragments are the expected digestion pattern from both SS1 and pTSIIDT, which contains the ICP4 gene (arrowheads). Twenty µg DNA of each sample was digested over night at 37°C and resolved in 1% agarose gel. Lane 1:HSV-1/NotI; Lane 2:d120/NotI; Lane 3:SS1/NotI; Lane4: pTSIIDT/NotI; Lane5:DNA marker (from top to bottom: 23, 9.4, 6.6, 4.4, 2.3, 2.0 and 0.5 Kb).</p

Mesothelin silencing reduces proliferation rate of pancreatic and ovarian cancer cells.

<p>(A)Mesothelin protein was detected in pancreatic cancer cell lines, Panc1, Miapaca2 and Bxpc3 and ovarian cancer cells Skov3 and Ovcar3. NIH3T3 and Huvec cells which are void of mesothelin were used to prove the specificity of mesothelin antibody. (B)Skov3 cells had reduced proliferation at day 3 post-electroporation with anti-mesothelin siRNA to about 50% of negative control. Callout panels show the density of cell at each time-point. (C–D)Two pancreatic cancer cell lines, Bxpc3 and Miapaca, were tested for the outcome of silencing mesothelin on their proliferation. In both cases a significant loss of proliferation was observed, however for Bxpc3 the decline initiates at later time points as compared with Miapaca cells. For both cells, once again, a rebound to higher proliferation rates is observed at longer time-points due to the clearance of siRNA from cells. Callout panels show the density of cell at each time-point.</p

Silencing MSLN confers inhibited cell proliferation and invasion.

<p>(A) Ovca429 cell were infected with MSLNmiR3 or Negative Control virus at MOI∼30. Cell proliferation was assessed at the indicated time-points. (B) Photographs of Ovca429 cells expressing EmGFP post-infection at the indicated time-points. 10× objective was used for taking pictures at day 10 and 20× was used at day 20 in order to focus on morphological changes of cells. (C) Left panel: Three-day post infection, Ovca429 cells were transferred and cultured in Boyden assay invasion chambers for 21 hrs. Cells were washed, fixed, and cell nuclei were stained by DAPI. Representative fields from each group were then counted for the number of nuclei and averages were calculated and compared (p<0.05). Numbers of cells invaded for the negative control virus was considered as 100%. Right panel: Photographs of invaded cells nuclei in MSLNmiR3 and negative control virus treated groups. The bar shows 100 µM in length.</p

Gene specific silencing of mesothelin reduces proliferation of mesothelioma cells.

<p>(A)Anti-mesothelin siRNA was designed to a middle sequence position in mesothelin mRNA. (B)Once electroporated with anti-mesothelin siRNA, the expression levels of mesothelin was significantly reduced in H2373 cells. Negative control siRNA did not cause such reduction. Lower panel shows the results of band-densitometry comparing the intensity of mesothelin expression upon electroporation of H2373 cells with siRNA. (C)Anti-mesothelin siRNA did not affect the expression levels of β-actin, a house-keeping protein, as an evidence for the specificity of this anti-mesothelin siRNA for its target. (D) Proliferation rate of H2373 cells is significantly (p<0.05) reduced at 48 hours post-electroporation to 40% of the values for negative control treated cells. A rebound to higher proliferation rates is observed due to clearance of siRNA from cells at later time points in harmony with our previous studies. Callout panels show the density of cells in each group of the study at 48 hours post-electroporation. (E) NIH3T3 cells are void of mesothelin and their proliferation rate is not affected by exposure to anti-mesothelin siRNA (mouse). Callout panels show the density of cells at 48 hour post-electroporation.</p