1. Volume 15, Issue 4, Pages 772-781 (April 2007)
Replication-deficient Adenovirus Induces Host Topoisomerase I Activity: Implications for Adenovirus-mediated Gene Expression 
Gideon Zamir, Evelyne Zeira, Andrew E Gelman, Abraham Shaked, Kim M Olthoff, Ahmed Eid, Eithan Galun 
Molecular Therapy 
Volume 15, Issue 4, Pages (April 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Adenovirus enhances topoisiomerase I activity in the liver of adenovirus-infected rats. (a) Nuclear protein extracts from livers of AdlacZ-infected (Infected) or non-infected (Non-infected) rats were incubated with plasmid DNA and, after de-proteinization, the mixtures were analyzed by gel electrophoresis together with intact (Native) and linearized (Linear) plasmids as markers, as described in Materials and Methods. Partially relaxed topoisomers of the plasmid, evident only in the mixture incubated with the infected extract, migrate mostly between the nicked-circular (NC) and linear (L) forms of the plasmid. SC, supercoiled plasmid. (b) Addi-tion of camptothecin (CPT) inhibits, in a dose-dependent manner, plasmid relaxation by the nuclear protein extract from adenovirus-infected rats. The results shown are representative of experiments with four animals in each group.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Topoisomerase I inhibition enhances transgene expression in HepG2 cells. Cells were infected with AdlacZ or AdGFP encoding for β-galactosidase or green fluorescent protein. Transgene expression was compared between topo-tecan-treated and untreated cells 24 hours after infection. Full view of wells with AdlacZ-infected cells without (a) or with (b) topotecan treatment. Same cultures viewed at ×20 magnification, untreated (c) or topotecan-treated cells (d). Fluorimetric imaging of cells infected with AdGFP without (e) or with (f topotecan treat-ment. Conditions were as described in Materials and Methods.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Quantification and characterization of topotecan-induced stimulation of transgene expression. (a) Quantitative analysis of β-galactosidase (β-gal) activity in HepG2 cells 24 hours after AdlacZ infection and 2- or 4-hour exposure to increasing concentrations of topotecan, added immediately after infection. Columns, mean; bars, ± SD. (b) Quantitative analysis of β-gal enzymatic activity in cells after infection with AdlacZ and treatment with hy-droxyurea or non-toxic concentrations of the topoisomerase II inhibitor etoposide. Columns, mean; bars, ± SD. (c) Cell viability at 1-50 μM topotecan or 100 μM hydroxyurea was determined using the MTT assay. Columns, mean; bars, ± SD. (d) Real-time polymerase chain reaction (PCR) analysis of the effect of 10 μM topotecan on viral DNA content in HepG2 cells 4 and 24 hours after AdlacZ infection. Cell wells, four per time point, were pooled before DNA extraction for collective analysis. (e) Flow cytometric analysis of the effect of topotecan treatment on cell-cycle progression. The cells were collected 12 hours after AdlacZ infection and 4-hour exposure to 10 μM topotecan or phosphate-buffered saline, added immediately after infection. The cells were stained with propidium iodide and analyzed for DNA content by flow cytometry. The nearly overlapping distribution of topotecan-treated and untreated cells indicates that under the conditions tested, topotecan had no meaningful effect on cell-cycle progression.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Effect of topotecan on transgene expression in non-hepatic cells. CT26 (colon cancer) (a, b) and PC3 (pros-tate cancer) (c, d) cells were infected with AdlacZ. Transgene expression was compared between topotecan-treated (b, d) and untreated cells (a, c) 24 hours after infection. Cultures were viewed at ×20 magnification. Conditions were as de-scribed in Materials and Methods.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Topotecan enhances hepatic transgene expression after systemic adenovirus infection. Mice were infected with 108 plaque-forming units of AdlacZ or AdGFPluc by tail vein injection followed by intravenous administration of 10 mg/kg topotecan or phosphate-buffered saline. Transgene expression in the liver was assessed 24 hours later. For detection of β-galactosidase expression, whole-mount 2-mm liver sections from untreated (a) or topotecan-treated (b) mice were subjected to X-gal staining. Insets show histological analysis of liver sections following hematoxylin and eosin staining. Blue staining marks β-galactosidase-expressing hepatocytes. (c, d) Green fluorescent protein (GFP) expression in whole mice viewed with a charge-coupled device camera 24 hours after systemic AdGFPluc infection, with (c) or without (d) topo-tecan treatment. Green fluorescence indicates GFP expression in the liver. Insets show a direct view of green fluorescence in the liver after skin incision. (e, f) Immunohistochemical analysis of luciferase expression in liver sections 24 hours after systemic AdGFPluc infection, without treatment (e) or with topotecan treatment (f). Similar results were obtained with three other mice.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
The effect of repeated topotecan administration on the course of luciferase accumulation and enzymatic activity in mouse liver after systemic infection by AdGFPluc. Mice (four per group) were treated with a single dose (20 mg/kg) of topotecan at the time of systemic AdGFPluc infection or with a second dose 24 hours later. Luciferase activity in topotecan-treated and untreated animals was monitored at days 1, 3, and 8 after infection by whole-body chemiluminescence analyses (a, b). Columns, mean; bars, ± SD. Levels of the liver-specific enzyme markers GOT (glutamic-oxaloacetic transaminase) and GPT (glutamic-pyruvic transaminase) determined in the serum at the same time points (c) indicated that the drug had no meaningful toxic effect under the conditions tested.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Topoisomerase I inhibition increases adenovirus-mediated gene expression in tumors. Human tumors: slices of 2-4 mm from co-lon cancer (a, b) or liver metastases (c, d) from the same patient were infected ex vivo with AdlacZ with or without the presence of topotecan. β-Galactosidase expression was assessed 24 hours after infection by whole-mount X-gal staining. Blue staining signifies β-galactosidase expression. Mouse tumors: BALB/c mice (four per group) harboring subcuta-neous CT26 colon cancer nodules were injected with AdGFPluc intra-tumorally followed immediately with intra-tumoral injection of 20 mg/kg topotecan or saline. In vivo luciferase expression was monitored by whole-body chemiluminescence 24 hours after infection.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions