We used four human STS cell lines, RD-ES (Ewing sarcoma), SK-ES-1 (Ewing sarcoma), HT1080 (fibrosarcoma), and NMS-2 (malignant peripheral nerve sheath tumor, MPNST). RD-ES, SK-ES-1, and HT1080 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). NMS-2 cells were kindly provided by Dr. Hiroyuki Kawashima (Niigata University, Niigata, Japan). Cells were not cultured for more than 5 months following resuscitation. The authentification was not performed by the authors. RD-ES and NMS-2 were grown in RPMI-1640 medium; SK-ES-1 cells were grown in McCoy’s 5a medium; HT1080 cells were grown in Eagle’s Minimum Essential Medium. All media were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2.
The recombinant telomerase-specific replication-competent adenovirus OBP-301 (Telomelysin), in which the hTERT promoter element drives the expression of the adenoviral E1A and E1B genes linked by an internal ribosome entry site, was previously constructed and characterized [7-9].
Cell viability assay
RD-ES and SK-ES-1 cells were seeded at a density of 3 × 103 cells/well, and HT1080 and NMS-2 cells were seeded at a density of 1 × 103 cells/well on 96-well plates, 24 h before irradiation or OBP-301 infection. In monotherapy, cells were irradiated at dosages of 0, 1, 2, 5, or 10 Gy using an MBR-1520R irradiator (Hitachi Medical Co., Tokyo, Japan). In combination therapy, cells were infected with OBP-301 at multiplicity of infections (MOIs) of 0, 1, 5, 10, or 50 plaque-forming units (PFU)/cell. Twenty-four hours after infection, cells were irradiated at dosages of 0, 1, 2, 5, or 10 Gy. Cell viability was determined on day 4 after irradiation using a Cell Proliferation Kit II (Roche Molecular Biochemicals Indianapolis, IN, USA) according to the manufacturer's protocol. The combined effect of OBP-301 and ionizing radiation was analyzed by calculating the combination index using the CalcuSyn software (BioSoft, Inc., Cambridge, UK). The computation of the combination index was based on the method of Chou .
Western blot analysis
SK-ES-1 and HT1080 cells (1 × 105 cells), seeded in a 100-mm dish, were prepared for protein extraction. Cells were treated with OBP-301 at the indicated MOIs for 48 h and/or were irradiated at the indicated dosages 24 h after infection. Cells were transfected with 10 nM MCL1 small interfering RNA (siRNA), or control siRNA (Applied Biosystems, Foster City, CA, USA) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 48 h and/or irradiated at the indicated dosages. Twenty-four hours after irradiation, whole cell lysates were prepared in a lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100) containing a protease inhibitor cocktail (Complete Mini; Roche Applied Science, Mannheim, Germany). To further assess DNA damage, SK-ES-1 cells (1 × 105 cells), seeded in a 100-mm dish, were infected with OBP-301 (10 MOI) and then irradiated at 1 Gy, 24 h after infection. Cells were harvested 0.5, 1, and 3 h after irradiation, and whole cell lysates were analyzed by Western blot for γH2AX expression.
Proteins were separated on 8–15% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Hybond-P; GE Health Care, Buckinghamshire, UK). Blots were blocked with Blocking-One (Nacalai Tesque, Kyoto, Japan) at room temperature for 30 min. The primary antibodies used were: mouse anti-Ad5 E1A monoclonal antibody (mAb) (BD Bioscience, Franklin Lakes, NJ, USA); mouse anti-γH2AX mAb (Merck Millipore, Billerica, Massachusetts, USA); rabbit anti-poly (ADP-ribose) polymerase (PARP) polyclonal antibody (pAb), rabbit anti-MCL-1 mAb, and rabbit anti-E2F1 mAb (Cell Signaling Technology, Danvers, MA, USA); and mouse anti-β-actin mAb (Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies used were: horseradish peroxidase-conjugated antibodies against rabbit IgG or mouse IgG (GE Healthcare). Immunoreactive protein bands were visualized using enhanced chemiluminescence (ECL Plus; GE Healthcare).
SK-ES-1 cells, seeded on tissue culture chamber slides, were infected with OBP-301 (5 MOI) for 48 h. Cells were irradiated at 1 Gy, and then 30 min, 1 h, or 3 h following irradiation, cells were fixed with chilled 1% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min on ice. The slides were subsequently incubated with primary mouse anti-γH2AX mAb (Millipore) for 24 h. After washing three times with PBS, slides were incubated with the secondary FITC-conjugated antibody against mouse IgG (Zymed Laboratories Inc., South San Francisco, CA, USA) for 30 min on ice. The slides were further stained with ProLong® Gold antifade reagent with DAPI (life technologies Co., Carlsbad, CA, USA) and then analyzed under a confocal laser microscope (FV10i; Olympus Co., Tokyo, Japan).
In vivo subcutaneous human STS xenograft tumor models
Animal experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of Okayama University School of Medicine. SK-ES-1 (5 × 106 cells/mouse) and HT1080 (3 × 106 cells/mouse) cells were subcutaneously inoculated into the flanks of 5- to 6-week-old female BALB/c nu/nu mice (CLEA Japan, Tokyo, Japan). When tumors reached 5 to 7 mm in diameter, the mice were irradiated at a dosage of 1 Gy/tumor (SK-ES-1) or 3 Gy/tumor (HT1080) every week for one or three cycles starting at day 0. During irradiation, mice were placed in a prone position, using custom-made holders that contain lead collimators to shield the upper half of the animal. OBP-301 at a dose of 1 × 108 PFU/tumor or PBS was injected into the tumors every week for one or three cycles. The perpendicular diameter of each tumor was measured every 3 days, and tumor volume was calculated with the following formula: tumor volume (mm3) = a × b2 × 0.5, where a is the longest diameter, b is the shortest diameter, and 0.5 is a constant to calculate the volume of an ellipsoid. At the end of the experiment, euthanasia was performed by isoflurane inhalation.
Tumors were fixed in 10% neutralized formalin and embedded in paraffin blocks. Sections were stained with hematoxylin/eosin. Sections were also prepared for immunohistochemical examination using mouse anti-γH2AX mAb (Millipore) and rabbit anti-Ki67 mAb (Abcam, Cambridge, UK). Immunoreactive signals were visualized using 3,3’-diaminobenzidine (DAB) solution (Nichirei Bioscience, Tokyo, Japan), and nuclei were counterstained with hematoxylin. All sections were analyzed under a light microscopy.
Sections were deparaffinized and put into 3% hydrogen peroxide for 10 min at room temperature. After nonspecific binding sites were blocked, the sections were incubated for 60 min at 37°C with terminal deoxynucleotidyltransferase mediated dUTP nick end labeling (TUNEL; Roche Applied Science, Penzberg, Germany) and stained with DAB solution. Finally, all sections were counterstained with hematoxylin. Sections were rinsed with PBS after every step.
Data were expressed as mean ± SD. Differences between groups were examined for statistical significance with the Student's t test. P values < 0.05 were considered statistically significant.