Figure 3.

Effect of oncolytic herpes simplex virus, poly(ADP-ribose) polymerase inhibitor (PARPi), and combination on DNA damage responses and cell cycle. A) PARPi-sensitive MGG4 (left) and PARPi-resistant BT74 (right) cells were treated with olaparib (O; 10 µM for MGG4 and 30 µM for BT74) or vehicle (mock) for 48 hours and then mock-infected or infected with MG18L (M) or G47Δ (G; MOI = 1) or in combination (O + M, O + G). Cells were harvested for immunoblotting at 30 hours after infection. β-actin as loading control. B) Effect of MG18L on homologous recombination. DR-GFP-transduced U2OS cells were transfected with a plasmid expressing I-SceI, and 24 hours later infected with MG18L (1MOI) or mock (PBS), followed 24 hours later by analysis of GFP-positive cells with fluorescence (left) and phase-contrast microscopy (middle). X-gal staining performed at 16 hours after MG18L infection (right), showing that almost all the cells were infected. Scale bars = 1 μm for GFP and phase contrast and 5 μm for X-gal. C) Representative fluorescence-activated cell sorting (FACS) analysis of GFP-positive cells (gated right quadrant, percent positive) from mock (top), MG18L infected (middle), and control nontransfected (lower). Total of 1X 10⁵ cells were analyzed for each sample. D) Quantification of GFP-positive cells analyzed by FACS as in (C) from three independent experiments. Data represented as mean ± SD; P = .002 (two-sided unpaired t test). E) Cell cycle analysis of treated MGG4 (left) and BT74 (right). Cells were treated as indicated with olaparib (3 μM for MGG4 and 30 μM for BT74) and/or MG18L (MOI = 0.5) and cell cycle phases determined after 24 hours by FACS. Values are the mean of three independent experiments and represented as mean ± SD. *P < .01; **P < .001; ∞P< .0001. P values of .01 or greater are not indicated (multiple comparisons test, Tukey). In MGG4: mock vs olaparib for G2/M, P = .004. In BT74: mock vs MG18L and olaparib vs Ola+MG18L for G1, P = .005; mock vs Ola+MG18L for S, P = .002. All statistical tests were two-sided. Ola = olaparib; PARP = poly(ADP-ribose) polymerase.