1. Volume 16, Issue 6, Pages 1033-1040 (June 2008)
Novel Cancer Antiangiotherapy Using the VEGF Promoter-targeted Artificial Zinc-finger Protein and Oncolytic Adenovirus 
Yoon-A Kang, Hyun-Chul Shin, Ji Young Yoo, Joo-Hang Kim, Jin-Soo Kim, Chae-Ok Yun 
Molecular Therapy 
Volume 16, Issue 6, Pages (June 2008)
DOI: /mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Schematic representations of the adenoviral (Ad) vectors used in this study. All indicated adenoviral vectors were derived from full-length adenoviral genomes cloned and manipulated in Escherichia coli as bacterial plasmids. (a) E1A-deleted replication-incompetent Ads. Ad-ΔE1-GFP expresses green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter; Ad-ΔE1-KOX expresses F435-KOX in the E3 region. (b) E1A-expressing oncolytic Ads. Ad-ΔB7 contains mutated E1A, but lacks E1B 19 and 55 kd; Ad-ΔB7-KOX expresses F435-KOX in the E3 region. Ad, adenovirus; ITR, inverted terminal repeat.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Quantitative enzyme-linked immunosorbent assay (ELISA) showing efficient inhibition of vascular endothelial growth factor-A (VEGF-A) expression by Ad-ΔE1-KOX. Cells were infected with either Ad-ΔE1-GFP or Ad-ΔE1-KOX at different multiplicities of infection (MOIs), and VEGF-A expression was measured in the culture medium 72 hours after transduction by conventional ELISA kits. Ad, adenovirus; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Effect of Ad-ΔE1-KOX on human umbilical vein endothelial cell (HUVEC) tube formation. HUVECs were plated on Matrigel-coated plates at a density of 2 × 105 cells/well and then incubated for 18–20 hours with the conditioned media of U343 and U87MG cells transduced with Ad-ΔE1-GFP or Ad-ΔE1-KOX. (a) Representative photographs of tube formation (×40). (b) Quantitative analysis of tube formation. Quantification of tube formation was carried out by measuring the area covered by the tube network using an optical imaging technique in all four quadrants of the well. The bar graph represents mean ± SE with 3 wells/treatment condition, *P < 0.05, **P < Ad, adenovirus; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Effect of Ad-ΔE1-KOX on vessel sprouting ex vivo. Aortas in Matrigel were treated with the conditioned media of U87MG cells transduced with Ad-ΔE1-GFP or Ad-ΔE1-KOX at an multiplicity of infection of 50 for 72 hours. The aortas were then cultured for 7 days and stained with Diff-Quick. (a) Representative photographs are shown from an experiment with five replicates per group (×40). (b) Quantitative evaluation of vessel spouting upon treatment with Ad-ΔE1-GFP or Ad-ΔE1-KOX. The vessel sprouting index was presented as mean ± SE (n = 7), *P < 0.05 versus phosphate-buffered saline treatment; **P < versus Ad-ΔE1-GFP treatment. Ad, adenovirus; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Semiquantitative assessment of oncolytic adenovirus (Ad)-mediated cytopathic effect. Cells were infected with Ad-ΔE1-GFP, Ad-ΔB7, or Ad-ΔB7-KOX at the indicated multiplicities of infection (MOIs) and cell killing was allowed to proceed for 4–9 days, followed by crystal violet staining to detect live cells. Each cell line was tested at least three times, and the data shown are from representative experiments. GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Characterization of oncolytic adenovirus (Ad) expressing F435-KOX. U87MG cells were infected with Ad-ΔB7 or Ad-ΔB7-KOX at an multiplicity of infection (MOI) of 1. At various time-points following infection, the supernatant and cell fraction were recovered and the viruses were extracted. (a) Kinetics of viral replication. Titers of released viruses were determined by limiting dilution assay on 293 cells. Each data point represents the mean ± SE of duplicate samples. Black circles, cells infected with Ad-ΔB7; white circles, cells infected with Ad-ΔB7-KOX. (b) vascular endothelial growth factor (VEGF) quantification. VEGF production in culture supernatants was measured by enzyme-linked immunosorbent assay. Each value represents the mean ± SE of duplicate samples *P < 0.05, **P < PFU, plaque forming units.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Enhanced induction of apoptosis by expression of F435-KOX. (a) The subG1 population quantified by flow cytometry after propidium iodide staining (three experiments). The subG1 apoptotic population is expressed as a percentage of total cells. Increased subG1 fractions were found in U87MG cells infected with Ad-ΔB7-KOX as compared with Ad-ΔB7. *P < 0.05 versus Ad-ΔB7 infection and **P < versus untreated control. (b) Effect of F435-KOX expression on the induction of apoptosis as measured using the terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) assay. At 48 hours after treatment with Ad-ΔB7 or Ad-ΔB7-KOX at an multiplicity of infection of 1, apoptotic cells were detected by TUNEL assay. Brown staining indicates positive staining for DNA strand breakage. Representative Fields are shown. Original magnification = ×200. The mean percentage of TUNEL-positive cells induced by Ad-ΔB7 or Ad-ΔB7-KOX *P < 0.05 versus Ad-ΔB7 infection and **P < versus untreated control. Ad, adenovirus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

9. Figure 8
Antitumor effect of F435-KOX-expressing adenoviruses (Ads). Suppressions of the growth of established tumors (a) and increased survival (b) following intratumoral injection with F435-KOX-expressing Ads. U87MG glioma xenograft tumors were grown subcutaneously in the abdomen of nude mice, and were subsequently injected with phosphate-buffered saline (PBS) (white squares), Ad-ΔE1-GFP (black triangles), Ad-ΔE1-KOX (white triangles), Ad-ΔB7 (black circles), or Ad-ΔB7-KOX (white circles). *P < Tumor volume was monitored over time (days) after treatment with adenoviruses (Ads). The percentage of surviving mice was determined by monitoring the death of the mice. Tumor size over 2,500 mm3 was regarded as death. (c) Histological assessment of angiogenesis in the tumor tissues treated with F435-KOX-expressing Ads. Ten days following final Ad administration, mice were sacrificed and the tumor tissues were harvested. Paraffin-embedded tumor sections were stained with hematoxylin and eosin (H&E). These sections are representative of treated mice from four independent experiments. Tumor tissues were also stained with an antibody against vascular endothelial growth factor (VEGF) or platelet endothelial cell adhesion molecule-1 (PECAM-1) and were hematoxylin counter stained. Brown staining indicated positive staining for VEGF and endothelial cells. Terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) staining of apoptotic cells in tumor tissues. Original magnifications = ×200. (d) Quantification of vessel numbers in tumor tissues. The data are presented as mean (n = 3) ± SE, *P < 0.05, **P < GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions