Adenoviral-delivery of p35 augments CPA bystander activity of Adeno-2B6-infected cultures. A) Immunostaining of CYP2B6 protein (blue) and TUNEL staining of apoptotic cells (dark brown) in populations of U251 cells infected for 48 hr with Adeno-2B6 (left photos) or Adeno-2B6/p35 (right photos). Following CPA treatment for 48 hr, apoptotic cells occurred more frequently in the Adeno-2B6/p35-infected culture as compared to the Adeno-2B6-infected culture. In addition, a greater fraction of the CYP2B6-expressing cells (blue) were apoptotic (black arrows) in the Adeno-2B6-infected cultures (34 out of 80 cells (42.5%) versus 11 out of 80 cells (13.8%) for Adeno-2B6/p35). Pink arrows: CYP2B6-expressing cells that are non-apoptotic. B) CPA-induced killing of 9L/lacZ bystander cells, assayed by colony formation activity. A mixed population comprised of 60% U251 cells (readily infectable by adenovirus; serve as P450 prodrug-activating "factory" cells) and 40% 9L/LacZ cells (uninfected at the adenovirus MOI used; serve as bystander cells) was infected for 48 hr by either Adeno-2B6 or Adeno-2B6/p35 at MOI 25. Cells were treated with 1 mM CPA using two different schedules, '8 hr × 2' (two 8 hr CPA treatments, with a 40 hr drug-free period between) or '24 hr × 2' (two 24 hr CPA treatments, with 24 hr drug-free between), modeled based on . The decrease in 9L/lacZ colony formation was 93% and 100% (8 hr × 2 and 24 hr × 2, respectively) in the Adeno-2B6/p35-infected cultures vs. 85% and 94% in the correspondingly treated Adeno-2B6 cultures. Unpaired t-tests were used to determine statistical significance: *: p < 0.05, **: p < 0.001. Data is represented as normalized 9L/lacZ colony formation mean ± SD between three different seeding densities.