Cell lines and culture
HCT-8, HCT116, HT-29 (human colon cancer cell lines) and GES-1 (normal human cell line) were obtained from the Shanghai Cell Collection, Chinese Academy of Sciences. The HEK293 cell line (human embryonic kidney cells containing the E1A region of the adenovirus) was obtained from Microbix Inc. (Ontario, Canada). HEK293, GES-1, and HCT-8 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone). HCT116 and HT-29 cells were maintained in RPMI 1640 (Invitrogen, CA, USA) supplemented with 10% FBS. Cells were maintained in a humidified incubator at 37°C with 5% CO2.
Plasmid construction and incorporation into adenoviral vectors
The original adenoviral shuttle plasmid that we used in this study was pDC315, and AD5 PBHGLOX1, 3CRE (Microbix Biosystems, Ontario, Canada). Mouse H19 enhancer exons 1 (258 bp) and 2 (360 bp) were amplified by PCR from mouse genomic DNA and then linked as a single fragment by PCR. The enhancer was cloned into the pDC315 plasmid using restriction endonucleases Xbal and EcoRI. Subsequently, mouse DMD exons 1–2 (429 bp), 3 (207 bp) and 4 (156 bp) were also amplified by PCR from genomic mouse DNA and then linked as a single fragment by PCR. The DMD was cloned downstream of the enhancer using restriction endonucleases EcoRI and NheI. The mouse H19 promoter (302 bp) was amplified by PCR from mouse genomic DNA using the following primers: forward, 5’ GCGCTAGCCCACCGTTCTATGAAGGGCTTC 3’ (containing a NheI site) and reverse, 5’ AAGGATCCTCATCAGCGCCCATCTCTAGCC 3’ (containing a BamHI site), and then cloned downstream of the DMD using restriction endonucleases NheI and BamHI. The human adenovirus E1A sequence (1013 bp) was amplified by PCR from a TOP-K plasmid, which was kindly provided by Dr. Ji-Fan Hu (Stanford University Medical School), using the following primers: forward, 5’ CCCGGATCCGGGCCCTATGAGACATATTATCT 3’ (containing a BamHI site) and reverse, CGCGTCGACCGCAATCACAGGTTTACACCTTA 3’ (containing a SalI site).
The EGFP reporter gene from the pEGFP-C1 vector (Clontech, Mountain View, CA, USA) and the E1A gene were then inserted downstream of the H19 promoter using restriction endonucleases BamHI and SalI to construct pDC315-enhancer-DMD-H19-EGFP and pDC315-enhancer-DMD-H19-E1A. Inserts were confirmed by DNA sequencing. The adenovirus Ad315-E1A was constructed by homologous recombination techniques using pDC315-enhancer-DMD-H19-E1A and the adenovirus packaging plasmid PBHGLOX1, 3CRE in HEK293 cells with Lipofectamine 2000 (Invitrogen Life Technologies, CA, USA). A standard replication-deficient adenovirus, Ad315-EGFP, was constructed by cotransfection of the adenovirus shuttle vector containing EGFP with an E1A/B-deleted adenoviral backbone vector.
Adenoviruses were plaque purified, propagated in HEK293 cells, and purified again by a CsCl gradient according to standard techniques. Functional particle titers of all adenoviruses were determined by a plaque assay using HEK293 cells. The positive control adenovirus H101 was kindly provided by Dr. Sheng-Fang Ge (Shanghai Jiao Tong University School of Medicine).
Cells were seeded in 96-well plates at a density of 10,000 cells/well for a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, or in 6-well plates at a density of 1000,000 cells/well for RT-PCR and flow cytometric analysis. Then, cells were incubated with various concentrations of Ad315-EGFP, Ad315-E1A, and H101 in serum-free DMEM at 37°C for 90 min. After incubation, serum-free DMEM containing the viruses was replaced with normal growth medium. The infected cells were maintained at 37°C until use in assays.
EGFP and E1A expression analyses
EGFP expression was examined at 24 h after infection with adenoviral vectors (10 PFU/cell) under an Axioskop 2 microscope (Carl Zeiss, Oberkochen, Germany) with a fluorescent filter set (excitation 450–490 nm). E1A mRNA expression was determined by RT-PCR. Total RNA was extracted using Trizol (Invitrogen Life Technologies) according to the manufacturer’s instructions. First strand cDNA synthesis was performed in a total volume of 25 μl containing 2 μg RNA, 0.5 μg primer and 200 U M-MLV reverse transcriptase. cDNA was subsequently amplified in 50 μl reaction volumes containing 0.4 μmol/l of each E1A primer and 1.25 U Taq DNA polymerase (TaKaRa, Dalian, China). The amplification conditions were pre-denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 40 s, 60°C for 40 s and 72°C for 60 s, and a final extension of 72°C for 7 min. PCR products were electrophoresed on a 1% agarose gel containing ethidium bromide and then visualized under UV light.
Western blotting to detect E1A protein expression
Cells were harvested and lysed by three cycles of freeze/thawing at −80°C. Cell lysates were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Membranes were probed with a mouse monoclonal antibody against E1A (Abcam, Boston, MA, USA) and then a horseradish peroxidase-conjugated goat anti-mouse IgG. Proteins were visualized using Lumi-Light Western Blotting Substrate (Roche Molecular Biochemicals).
Cytotoxicity was assessed using an MTT assay that was performed as described previously using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan). Briefly, cells were seeded in 96-well plates at a density of 10,000 cells/well. Then, cells were infected with recombinant adenoviral vectors (10 PFU/cell). Cell growth and viability were assayed at 72 h after incubation with CCK-8 kit reagents by measuring absorbances at 450 nm in a microplate reader (Bio-Rad, Richmond, CA, USA). Each sample was assayed in quadruplicate, and experiments were repeated at least twice.
Quantitative evaluation of apoptosis was performed using flow cytometry after double staining with an annexin V-fluorescein isothiocyanate apoptosis detection kit to discrimination between early apoptotic (annexin V-positive) and necrotic ( annexin V/propidium iodide double-positive) cells. The cells (100,000 cells/well) were cultured in 6-well plates, and then infected with Ad315-E1A at 10 PFU/cell. Cell apoptosis was analyzed at 72 h after infection with Ad315-EFGP (10 PFU/cell) serving as a negative control.
Tumor xenografts were established by subcutaneous injection of 5 × 106 HCT-8 cells into the right flank of 4–6-week-old female athymic nude mice, which was approved by the Experimental Animal Center of University of Yangzhou, Yangzhou, China. The average tumor volume was measured by the formula: volume = (length × width2) / 0.5. When tumors reached approximately 100 mm3, xenografted mice were randomly divided into three treatment groups with eight mice in each group. The Ad315-E1A group received intratumoral injections of 1 × 108 Ad315-E1A viral particles three times every other day. Tumors injected with Ad315-EGFP viral particles at the same dosage served as a viral vector control, and those injected with PBS served as a negative control. The tumor size was measured by vernier calipers every 3 days. Mice were then euthanized by cervical dislocation at a predetermined interval of observation. Tumors were dissected out and stored in 40% formalin. For histological analysis, fixed tumors were embedded in OCT compound and then cut into 5–7 mm sections using a Cryocut microtome (Leica, Germany). Mouse survival was recorded in a separate experiment. Animal experiments were performed in accordance with institutional guidelines for animal care by Nanjing Medical University.
To immunohistochemically stain the E1A protein, the tumor and neighboring tissues were fixed in 10% formalin and embedded in paraffin for staining. Tumor or tissue sections were incubated at 4°C overnight with a mouse anti-human E1A antibody (Abcam, Boston, MA, USA) at a dilution of 1:50. Sections were rinsed in PBS-T (0.05% Triton X-100 in PBS), followed by incubation with a goat anti-mouse secondary antibody at a 1:500 dilution for 1 h at room temperature. Sections were subsequently incubated with streptavidin-horseradish peroxidase (BD Biosciences) and diaminobenzidine substrate to develop the colorimetric reaction. The number of E1A-positive cells was counted in five random fields at ×400 magnification under a light microscope and averaged. Only cells with distinct staining were counted. The positivity rate was used to grade the expression levels.
Apoptosis of tumor cells was detected using a TUNEL assay performed with an In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) following the manufacturer’s instructions. To stain apoptotic cells, tumor samples were fixed with 10% formaldehyde and paraffin-embedded sections were prepared. The number of TUNEL-positive cells was counted in five random fields at ×400 magnification under a light microscope, and the apoptosis index for each field was calculated as the percentage of TUNEL-positive cells relative to the total number of cells.
Experimental data were presented as the mean ± standard deviation (SD) and assessed using the Student’s t-test and one-way analysis of variance. Differences among the results of in vivo survival experiments were assessed by the Kaplan-Meier method. Results were considered statistically significant at p < 0.05.