Design and characterization of Adeno-2B6/p35. A) Schematic showing separate p35 and P450 expression cassettes introduced into Adeno-2B6/p35. The P450 cassette encodes CYP2B6 linked to P450 reductase cDNA via an internal ribosome entry sequence ('IRES') and was originally derived from Adeno-2B6 . B) Western blots showing CYP2B6 (top) and p35 (bottom) protein in 293 cells infected for 48 or 72 hr with either Adeno-2B6 and Adeno-2B6/p35/p35 at MOI 0, 10 or 20, as indicated. Importantly, the level of CYP2B6 protein was similar in cells infected with Adeno-2B6 and Adeno-2B6/p35. C) Functional activity of the CYP2B6-expressing adenoviruses, shown by HPLC analysis of 4-OH-CPA production by U251 cells infected with Adeno-2B6 or Adeno-2B6/p35 (MOIs 0, 3.75, 7, and 15; 48 and 72 hr post-infection) after incubation with 1 mM CPA for 4 hr (mean ± SD, n = 2).