Cell lines and reagents
CPA was purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) and RPMI 1640 medium were purchased from Invitrogen (Frederick, MD). Human tumor cell lines U251 (brain) and A549 (lung) were obtained from Dr. D. Scudiero (National Cancer Institute, Bethesda, MD). The rat 9L gliosarcoma tumor cell line was obtained from the UCSF Neurosurgery Tissue Bank (San Francisco, CA). Tumor cells were grown at 37°C in a humidified, 5% CO2 atmosphere in RPMI 1640 culture medium containing 5% FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml). The conditionally replicating adenovirus ONYX-017 , which contains a wild-type viral E3 region and an E1B-55 kDa gene deletion, was obtained from ONYX Pharmaceuticals (Richmond, CA).
Construction of Adeno-2B6/p35
Virus was constructed in the following three steps. (1) Subcloning of p35 into pORF, to create a p35 expression cassette: p35 cDNA was excised with EcoR I from pBluescript-p35, obtained from Dr. Thomas D. Gilmore (Boston University, Boston, MA), and cloned into pORF-MCS (InvivoGen, San Diego, CA) linearized with EcoR I. The resulting plasmid, pORF-p35, was digested with Bgl I to select for clones with the correct orientation. (2) Subcloning of p35 expression cassette into pShuttle-2B6-IHOR: To obtain a blunt-ended p35 expression cassette driven by a hEF1-HTLV promoter, pORF-p35 was digested with BfuA I then blunt-ended using Klenow enzyme. The linearized plasmid was then digested with Swa I. To clone the p35 expression cassette into pShuttle-2B6-IHOR, which contains CYP2B6 cDNA in linked to P450 reductase cDNA via an internal ribosome entry sequence , pShuttle-2B6-IHOR was first digested with Mlu I, dephosphorylated, and blunt-ended with Klenow enzyme. The p35 expression cassette and pShuttle-2B6-IHOR vector, both gel purified, were ligated to create pShuttle-2B6-IHOR-p35. (3) Subcloning of 2B6-IHOR-p35 into Adeno-X: To obtain the final recombinant adenoviral DNA, a 2B6-IHOR-p35 fragment was excised from pShuttle-2B6-IHOR-p35 by PI-Sce I/I-Ceu I double digestion followed by ligation with Adeno-X viral DNA linearized using the same enzymes. The reaction mixture was then digested with Swa I to eliminate non-recombinants. Clones containing the 2B6-IHOR-p35 fragment were identified by PCR using CYP2B6 primers. Adenovirus was then generated following the manufacturer's instructions (Clontech, Mountain View, CA, USA). The recombinant Adeno-X-2B6-IHOR-p35 was digested with Pac I, and the linearized viral plasmid was transfected into low passage HK293 cells. A cytopathic effect was apparent after 10-14 days, at which point Adeno-2B6/p35 virus was isolated, amplified in HEK293 cells, purified, and quantified using the Adeno-X Rapid Titer kit (Clontech Labs), as described in the manufacturer's protocol .
qPCR was performed as described  using gene-specific primers for quantification of CYP2B6 (sense: 5'-ACTGCTCTCCATGACCCACACT-3' and antisense: 5'-CGATGCCTTCACCAAGACAAA-3') and p35 cDNA (sense: 5'-TCGACGAACGCAACGACTAC-3' and antisense: 5'-CTTGGTTGCTGCCGTTCTC-3').
Cell extracts (20 μg) prepared in lysis buffer (20% glycerol, 1% Triton X-100, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na4P2O7, 1 mM DTT, 1 mM Na3VO4, 1 mg/ml leupeptin, and 1 mg/ml pepstatin) were analyzed by Western blotting. CYP2B6 protein was detected using a monoclonal rabbit anti-P450 2B6 antibody (1:3000 dilution; Gentest Corp., Woburn, MA), and p35 protein was detected using polyclonal rabbit anti-p35 antibody (1:3000 dilution; Biocarta, CA). Goat anti-rabbit HRP-conjugated secondary antibody (1:3000 dilution) was used for both primary antibodies (Amersham). Blots were visualized with enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham Pharmacia Biotech) exposed to Kodak X-OMAT blue film XB-1, and scanned to verify equal expression of CYP2B6 in tumor cells infected with Adeno-2B6 and Adeno-2B6/p35.
Quantification of cellular 4-OH-CPA production
U251 cells were plated at 100,000 cells/well with 1 ml medium/well in a 12-well plate. 24 hr later, cells were infected with Adeno-2B6 or Adeno-2B6/p35 at multiplicities of infection (MOIs) of 0, 3.75, 7, and 15. Cellular CPA 4-hydroxylase activity was determined 48 hr and 72 hr post-infection by 4-OH-CPA released into fresh culture medium containing 1 mM CPA over a 4 hr period. 5 mM semicarbazide was included in the medium to trap and stabilize the initial 4-OH-CPA metabolite. An aliquot of medium (0.5 ml) was removed from each well and snap frozen in liquid nitrogen and stored at -80°C until processing and HPLC analysis as previously described . Cells remaining on the plate were washed with 1X PBS and stained with crystal violet (A595) to normalize 4-OH levels to cell content for calculation of cellular CPA 4-hydroxylase-specific activity (nmol 4-OH-CPA/ml media/A595). HPLC calibration curves were generated using standard curves of 4-OH-CPA metabolite prepared using 4-OOH-CPA dissolved in cell culture medium .
Caspase activity assay
Caspase-3/7 activities were measured using the Apo-ONE homogeneous caspase-3/7 assay (Promega, Madison, WI). Cells were plated in a 96-well plate at a density of 1,000 cells/0.1 ml/well. 24 hr after cell seeding, cells were infected with Adeno-2B6 or Adeno-2B6/p35 at MOIs specified in each figure. 48 hr after viral infection, medium was aspirated and 1 mM CPA was added in a vol of 60 μl/well. 48 hr after beginning drug treatment, caspase-3/7 activity was assayed by adding an equal volume (60 μl) of homogeneous caspase-3/7 reagent to each well, and the plate was incubated for 2 hr at room temperature with constant shaking at 300 rpm. Fluorescence was measured with an ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA) using an allelic discrimination method with rhodamine 110 as a reporter dye. The value of bin 7, which is the peak of the raw fluorescence curve, was used to indicate the activities of caspase-3/7.
Immunohistochemical analysis and TUNEL staining
For immunohistochemistry, cells were fixed for 20 min with 4% formaldehyde and permeabilized with 1% Triton-X100/1% sodium citrate for 5 min at 4°C. Blocking was performed using 2% normal goat serum for 1 hr at room temperature. Cells were stained with 1:2000 diluted mouse anti-human 2B6 monoclonal antibody (GenTEST, BD Biosciences, Cat. #458326) for 1 hr at room temperature. Secondary antibody incubation used HRP-conjugated anti-mouse IgG (1:500 dilution) at room temperature for 1 hr. DAB or VIP was used as the substrate for HRP. TUNEL staining was used to assay the effect of p35 on CPA-induced apoptosis, as follows. 6,000 U251 cells were seeded in two 4-well chamber slides. The next day, cells were infected with Adeno-2B6 or Adeno-2B6/p35 at MOIs of 0 and 25. 48 hr post-infection, 1 mM CPA was added to each well for 48 hr. Slides were then fixed with 4% paraformaldehyde and processed with the DeadEnd colorimetric TUNEL system (cat. no. G7360; Promega). For double staining, immunohistochemistry for CYP2B6, as above, was carried out following TUNEL staining.
Growth inhibition assay
To assay the effect of p35 on CPA-induced cell death, 15,000 U251 cells were plated in 24-well plates 24 hr prior to viral infection. The next day, cells were infected with either Adeno-2B6 or Adeno-2B6/p35 at MOIs 0, 2.5, 5, and 10 in a minimal volume of 200 μl for 3 hr, following which 1 ml of fresh medium was added/well. 48 hr after viral infection, either no drug or 1 mM CPA was added to the cells for 4 days. Cells were stained with crystal violet and quantified by measuring the 70% ethanol-eluted crystal violet (A595). To assay cisplatin and doxorubicin cell sensitivity, U251 cells were seeded in a similar fashion but were infected with either virus at MOIs 15 and 30 and then treated for 4 days at 0, 5, 10, 20, and 40 μM (cisplatin) or at 0, 20, 40, 80, and 160 nM (doxorubicin).
Since 9L cells are virtually uninfectable by adenovirus at MOIs ≤100 (data not shown), 9L cells were used as uninfectable bystander cells. To assay bystander killing, U251 and 9L/LacZ cells were mixed at a ratio of 60:40 then plated at 150,000 cells/well of a 12-well plate. 24 hr later the cells were infected with either Adeno-2B6 alone, Adeno-2B6/p35 alone, Adeno-2B6 + ONYX-017, or Adeno-2B6/p35 + ONYX-017 at MOIs calculated based on the number of seeded U251 cells, as indicated in the figures. 48 hr after viral infection, mixed cells were treated with 1 mM CPA for either 8 or 24 hr. A second treatment with 1 mM CPA for 8 or 24 hr was applied to the cells 48 hr after the beginning of the first CPA treatment. 16 hr after the end of the second CPA treatment, cells were trypsinized, counted, and reseeded at various densities in 6-well plates to allow for the growth of individual colonies, as follows: drug-free control cells were replated in duplicate at 100, 200, and 400 cells/well; and CPA-treated cells were replated at 1,000, 2,000, and 4,000 cells/well. Cells were grown for 7-9 days, and then stained with X-Gal. LacZ-positive colonies containing ≥50 cells were counted. The efficiency of colony formation was then calculated .
ONYX-017 helper virus studies
To determine whether expression of p35 from Adeno-2B6/p35 interferes with the replicating virus helper effect or viral spread by ONYX-017, U251 and A549 cells were plated at 8,000 cells/well of a 24-well plate, and 24 hr later the cells were infected for 4 hr with either Adeno-2B6, Adeno-2B6/p35, Adeno-2B6 + ONYX-017, or Adeno-2B6/p35 + ONYX-017 (MOI 5 for Adeno-2B6 and Adeno-2B6/p35, and MOIs of 0, 1, or 3 for ONYX-017). Virus was then removed and fresh medium was added to each well. Cell supernatants were isolated on days 1, 2, 3, 4, 5, and 6 post viral infection, and qPCR was performed on aliquots to assay for viral presence using primer sets for CYP2B6 and p35 cDNA, as described above. Viral particle numbers were determined by normalization to a calibration curve generated by qPCR analysis of known viral dilutions. To assay the ultimate extent of p35 bystander activity augmentation via the viral helper effect, 7,000 U251 + 9L/lacZ cells (ratio of 40:60) were seeded in a 12-well plate. The following day, cells were infected with ONYX-017 (MOIs of 0, 1, and 3) in combination with either Adeno-2B6 or Adeno-2B6/p35 (MOIs of either 7.5 or 15, using MOI values calculated based on initial U251 cell numbers). After a 48 hr viral incubation, either no drug or 1 mM CPA was added to the cells for 48 hr. 48 hr later the cells in each well were trypsinized, counted and replated at densities of 1,000 and 3,000 cells in two wells of a 6-well plate. Colony formation then proceeded for 5-7 days, after which the cells were stained with X-gal and counted to determine the impact of p35 expression on bystander activity, as judged by a decrease in tumor cell colony formation.
Data is presented as mean ± SD based on either technical duplicates or triplicates, as indicated. In addition, to ensure that all findings were reproducible and representative, experimental results were confirmed in at least 2 or 3 independent sets of experiments.