ONXY-017 helper virus increases p35-enhanced bystander activity. Colony formation assay of re-seeded 9L/lacZ cells and U251 cells (initially seeded at a 60:40 ratio) following co-infection for 48 hr with ONYX-017 (MOI 0, 1, or 3) and either Adeno-2B6 or Adeno-2B6/p35 (MOI 7.5 or 15) followed by treatment with 1 mM CPA for 48 hr. 9L/lacZ bystander cells were X-gal stained and then quantified by A650 after elution of the stain with DMSO. ONYX-017 conferred helper effects for both Adeno-2B6 and Adeno-2B6/p35, increasing killing of 9L/lacZ bystander cells as a result of the increase in CYP2B6 expression, 4-OH-CPA production and ultimate cell killing. The decrease in 9L/lacZ colony formation, reflecting an increase in bystander activity, was substantially greater with Adeno-2B6/p35 as compared to Adeno-2B6.