1. Volume 123, Issue 2, Pages 608-618 (August 2002)
Gene therapy by intrahepatic and intratumoral trafficking of p53-VP22 induces regression of liver tumors 
Lars Zender, Reiner Köck, Matthias Eckhard, Bernd Frericks, Thomas Gösling, Thomas Gebhardt, Susanne Drobek, Michael Galanski, Florian Kühnel, Michael Manns, Stefan Kubicka 
Gastroenterology 
Volume 123, Issue 2, Pages (August 2002)
DOI: /gast
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2. Fig. 1
Transcriptional transactivation by p53wt and p53-VP22. (A) Transfection experiments with a reporter plasmid containing a luciferase gene linked to a p53-dependent promoter (prMin-RGC) were performed in p53-negative Hep3B hepatoma cells. Expression vectors for p53wt, p53-VP22, VP22-p53, and dominant-negative p53-249S were cotransfected as indicated. The C-terminal fusion protein p53-VP22 showed a 4-fold stronger transactivation capacity than p53wt and a more than 6-fold reduced susceptibility to repression by dominant-negative p53-249S. Transfection rates in Hep3B hepatoma cells were about 40% as shown by lacZ staining. (B) To rule out artifacts due to VP22-mediated protein trafficking, experiments were repeated in highly transfectable 293T HEK cells (transformed human embryonal kidney cells). After transfection of at least 90% of the cell layer, as shown by lacZ staining, significantly higher transactivation capacity of p53-VP22 compared with p53wt could be confirmed. The results shown represent the mean of 3 independent experiments.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Schematic representation of the expression cassettes in constructed adenoviral vectors used in this study. Recombinant adenoviruses were constructed using the AdEasy system. In the resulting vectors, expression of transgenes is driven by CMV promoters. Each vector contains a GFP expression cassette for convenient monitoring of infected cells.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
Efficient intercellular spread of p53-VP22 encoded by an adenoviral vector in normal liver. Balb/c mice (6–8 weeks old) received 1 × 109 pfu AdGFP/p53-VP22 or AdGFP/p53wt via tail vein. Livers were harvested 48 hours after infection for visualization of transduced cells using GFP fluorescence. Expression of p53 was monitored in the same sections by immunofluorescence. Using Ad-GFP/p53wt, every p53-overexpressing cell had been transduced by adenovirus. After infection with AdGFP/p53-VP22, more p53-positive than GFP-positive hepatocytes could be identified whereby primary expressing cells showed a significantly stronger fluorescence signal than secondary recipient hepatocytes. (A) Higher magnification of the sections shows a predominant nuclear localization of p53wt, whereas (B) the p53-VP22 protein shows a more cytoplasmic pattern of distribution in primary expressing hepatocytes and secondary recipient hepatocytes.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
VP22 mediates widespread intercellular transport of p53 in liver tumors in vivo. Subcutaneous Hep3B tumors were transduced with 5 × 109 pfu of AdGFP, AdGFP/p53wt, or AdGFP/p53VP22 by intralesional needle injection. Twenty-four hours after treatment, tumors were harvested and investigated for GFP immunofluorescence and p53 immunofluorescence. Patterns were identical in tumor nodules treated with AdGFP/p53, but a widespread VP22-mediated transport of p53 could be observed with AdGFP/ p53-VP22.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
In vitro transduction of hepatoma cells with AdGFP/p53wt or AdGFP/p53-VP22 results in p53- and p53-VP22–mediated apoptosis. (A) Hep3B hepatoma cells (homozygous deletion of p53 gene) and McA-RH7777 hepatoma cells (overexpressing a p53 mutant) were infected with either AdGFP/p53wt or AdGFP/p53-VP22 in a multiplicity of infection of 0.8 (0.8 viral particles per cell). Dead cells were detected by flow cytometry using propidium iodide staining. The portion of dead cells is indicated for each experiment. The data shown are representative of 3 independent experiments. (B) We repeated the experiments in Hep3B cells with annexin V staining, which detects earlier stages of apoptosis and may be a more sensitive method for detection of apoptosis. The data shown represent the mean fluorescence measured after administration of either AdGFP/p53wt or AdGFP/p53-VP22 in a multiplicity of infection of 0.4 or 0.8 particles per cell. The results shown represent the mean of 3 independent experiments.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
Adenoviral delivery of p53-VP22 leads to regression of liver tumors in vivo. A total of 1 × 106 Hep3B hepatoma cells were injected subcutaneously into the flanks of nude mice. Nude mice bearing established tumors were subsequently treated with an intratumoral injection of AdGFP, AdGFP/p53wt, or AdGFP/p53-VP22 at a dose of 5 × 109 pfu. Tumor size was measured at the indicated time points by direct calibration. Each graph represents the mean values of measured tumor size of 3 treated animals ± SEM.
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
Intra-arterial treatment of orthotopic hepatocellular carcinomas with AdGFP/p53-VP22 leads to significantly higher tumor regression compared with treatment with AdGFP/p53wt. (A) Tumor size of orthotopic hepatocellular carcinoma was monitored by MRI one day before and 10 days after intra-arterial gene therapy with AdGFP/p53wt or AdGFP/p53-VP22. All animals treated with AdGFP/p53wt showed progressive disease, whereas objective response rate in the group of animals treated with AdGFP/p53-VP22 was 50% (2 complete remissions and one partial remission). (B) Animals treated with AdGFP/p53-VP22 showed longer survival than animals treated with the control vector AdGFP/p53wt. Even the animals with progressive disease according to the World Health Organization criteria after treatment with AdGFP/p53-VP22 survived longer than the animals in the control group. Animals with complete remission on MRI showed long-term tumor-free survival for at least 16 weeks.
Gastroenterology  , DOI: ( /gast )
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9. Fig. 8
A single injection of 5 × 109 pfu AdGFP/p53-VP22 into the gastroduodenal artery of Buffalo rats leads to complete regression of liver tumor in a syngeneic orthotopic model of hepatocellular carcinoma. Transverse T1-weighted images before and after treatment of a solitary lesion in animal 2 are shown. (A) Before treatment, the tumor shows a maximum diameter of 0.5 cm and (B) a contrast enhancement after intravenous Magnevist application. (C) After treatment, an almost complete regression in tumor size can be detected. Only a star-like scar can be seen in upper part of C as a residuum of the tumor. (D) In addition, no contrast enhancement can be observed after treatment.
Gastroenterology  , DOI: ( /gast )
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10. Fig. 9
Intra-arterial injection of 5 × 109 pfu AdGFP/p53-VP22 leads to more homogeneous distribution of necrotic areas within the tumor compared with intra-arterial application of AdGFP/p53wt. Orthotopic liver tumors were harvested 4 days after intra-arterial gene therapy with either AdGFP/p53-VP22 or AdGFP/p53wt. Hemalaun-eosin staining of cryosections was performed. An AdGFP/p53-VP22–treated tumor showed broad areas of necrosis, whereas only small regions of necrosis could be observed within the AdGFP/p53wt–treated tumors. No substantial infiltration of mononuclear cells could be detected, indicating that immunologic mechanisms were less important for AdGFP/p53-VP22–mediated antitumor effects.
Gastroenterology  , DOI: ( /gast )
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