Figure 4

Effects of different combination treatments on cell survival, apoptosis and cell cycling: OVCAR-3 or SKOV-3 cells treated with docetaxel, carboplatin and PNP-GDEPT (with Fludara @ 1 μg/mL (2.7 μM)) either alone or in combination were analysed for cell survival by: Panel A . Clonogenic assay, Representative photographs show the crystal violet stained colonies of OVCAR-3 cells given different treatments (docetaxel (0.6 nM), carboplatin (40 μM) and PNP-GDEPT (Ad/CMV/PNP moi of 4 pfu/cell plus 1 μg/mL (2.7 μM) Fludara) either alone or in combination (in triplicate) in a 6 well plate. The graph and the values in the panel A represents the number of colonies/treatment group and represent mean (± SEM) of two independent experiments. Values were compared by One Way Anova using Dunnet's multiple comparison test. A P value < 0.05 was considered significant (*), however, exact P values for each data set are shown on the graphs. Panel B . Quantitative estimation of apoptosis in ovarian cancer cells (M30 CytoDEATH assay): SKOV-3 cells were treated with docetaxel (DOC) (3 nM), carboplatin (CAR) (20 μM) and PNP-GDEPT (Ad/CMV/PNP moi of 200 pfu/cell plus 1 μg/mL (2.7 μM) Fludara) either alone or in combination. Cells harvested 1 and 2 days post treatment were immunostained with M30 cytoDEATH antibody followed by flow cytometry. Graph shows the percent of M30 positive cells (represented apoptotic cell death) on different days post treatment with different modalities (alone or in combination). The statistical significance of data on the two days was determined by one-way Anova and using Tukey's multiple comparison tests. Overall P values and the significant differences between control, mono-, and di- and tri-combination treatments effects are displayed: * = P < 1.01, ** = P < 0.001, *** = P < 0.0001. Panel C . Cell cycle analysis, OVCAR-3 cells treated with different modalities (either alone or in combination; docetaxel (0.6 nM), carboplatin (10 μM) and PNP-GDEPT (Ad/CMV/PNP moi of 10 pfu/cell plus 1 μg/mL (2.7 μM) Fludara) either alone or in combination.) were assessed for cell cycle progression and apoptosis. Cells were harvested 48 h post treatment, RNA was digested and DNA was stained with propidium iodide. The histograms represent the fraction of cells in different phases of the cell cycle after different treatments as determined by flow cytometry. First histogram is representative of different phases of cell cycle in normal untreated cells. While data from one representative experiment is shown, numbers in each panel show the % distribution of cycling cells as mean ± SEM from three independent experiments.