Figure 3. All replication-defective E1A12S mutants sensitise prostate cancer cells to mitoxantrone and docetaxel except the AdE1A1104 virus.

A) Drug dose responses in each cell line were evaluated after infection with AdE1A12S, AdE1A1102, AdE1A1104, and AdE1A1108 mutants with AdGFP as negative control to determine changes in drug EC₅₀ values. All cell lines were infected at doses killing <10% of cells alone; PC-3 cells at 100 ppc (left panel), DU145 cells at 10 ppc (mid panel) and 22Rv1 cells at 2.5 ppc (right panel). Data represent averages ±SD, n = 4–5 independent experiments analysed by t-test comparing EC₅₀ values for each combination to that of drug alone, expressed as percentages, *p<0.05 and °p<0.01. B) EC₅₀ values for mitoxantrone were determined with and without simultaneous infection with viral mutants at 2.5, 10 and 100 ppc for 22Rv1, DU145 and PC-3 respectively, and with (grey bar) and without (black bar) the addition of the pan-caspase inhibitor zVAD-fmk at 25 µM. EC₅₀ values are expressed as percentages of mitoxantrone alone (Ctrl), averages ± SD, n = 3. C) Flow cytometry of cells infected with the AdE1A12S mutants or treated with mitoxantrone (50 nM) alone and in combination and analysed for tetramethylrhodamine uptake (TMRE) as an indicator of mitochondrial depolarisation and apoptosis induction. AdE1A12S, AdE1A1102, AdE1A1104, AdE1A1108, and AdGFP alone (solid arrow; all cell lines) and AdE1A12S, AdE1A1102, AdE1A1104 and AdE1A1108 in combination with mitoxantrone (dashed arrow; 22Rv1 cells). Data expressed as % apoptotic cells; percentages of cells that showed mitochondrial depolarisation, averages ± SD, n = 3. D) Cells infected with each mutant at 100 (PC-3) or 2.5 ppc (22Rv), mock infected and treated with or without mitoxantrone at 50 nM. Changes in cell cycle were analysed by flow cytometry at 24, 48 and 72 h after infection and drug treatment in PC-3 cells or after 48 h for 22Rv1 cells, one representative study (n = 3), *p<0.05 comparing G2/M-phase in combination treated vs mitoxantrone alone, °p<0.05 G2/M-phase for mitoxantrone vs mock treated.