1. Volume 16, Issue 10, Pages 1681-1687 (October 2008)
Dual Therapeutic Effects of Interferon-α Gene Therapy in a Rat Hepatocellular Carcinoma Model With Liver Cirrhosis 
Kai-Wen Huang, Yung-Chi Huang, Kuo-Feng Tai, Bo-Hua Chen, Po-Huang Lee, Lih-Hwa Hwang 
Molecular Therapy 
Volume 16, Issue 10, Pages (October 2008)
DOI: /mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Liver tumors with and without liver cirrhosis. The animals were divided into four groups (n = 50 each). They were given access to regular water only (control group) or to diethylnitrosamine (DEN) solutions (classical, A and B groups) in accordance with the protocols described in Materials and Methods. Three animals from each group were killed each week. Their liver weights, spleen weights, and body weights (BWs) were measured and expressed as (a) liver weight/BW and (b) spleen weight/BW. The ratios of liver weight/BW in groups A and B are significantly higher than those in the control and classical groups at week 12 [***P < 0.001, analysis of variance (ANOVA)] (as shown in a), whereas only group B had a ratio of spleen weight/BW significantly higher than those of the control and classical groups at week 12 (***P < 0.001, ANOVA) (as shown in b). Representative liver pictures of (c) group A animals and (d) group B animals at week 12 of DEN administration. These images indicate cirrhotic features in the livers of group B animals but not in those of group A animals.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Irreversible fibrosis induced by diethylnitrosamine (DEN) in group B animals. (a) Quantification of Sirius red staining. The formalin/paraffin sections of liver tissue free of tumor nodules from each group at the indicated time of DEN administration were stained with Sirius red. The percentages of Sirius red (+) areas were determined from 10 fields using the Digital Camera System and Image-Pro Plus software. *P < 0.05; **P < 0.005; ***P < 0.001, as compared to the control group. (b) Measurement of bile flow rates. Bile flow rate was measured at week 12, just before the animals were killed (n = 5 in each group). The animals were anesthetized with 80 mg/kg ketamine hydrochloride, a PE10 silicon tube was placed in the common bile duct, and bile was drained into an Eppendorf tube. The volume of bile accumulated in the tube was measured at 15-min intervals. **P < 0.005, as compared to the control group. (c) Measurement of hydroxyproline content. Liver collagen content was determined by quantifying the levels of hydroxyproline as previously described.34 A significant difference in this parameter was observed between group B and the other three groups. ***P < 0.001, analysis of variance (ANOVA). (d) Analysis of transforming growth factor-β (TGF-β) mRNA expression in the liver. Total RNA was isolated from the livers of group A and group B animals at the indicated time points of DEN administration, and analyzed for TGF-β expression using real-time quantitative reverse transcriptase PCR. TGF-β mRNA levels were normalized to those of β-actin mRNA. A significant difference was observed between groups A and B at week 12. *P < 0.05, ANOVA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Hepatocellular carcinoma induced by diethylnitrosamine (DEN). (a) Measurement of tumor burden. The livers obtained from the sacrificed animals at each time point were sliced into 5-mm sections. The numbers and sizes of all visible tumor nodules with diameters >3 mm were counted and measured. Tumor burden was expressed as the sum of the volumes of total tumor nodules. ***P < as compared to the control group. (b) Glutathione S-transferase, placental form positive [GST-P (+)] foci induced by DEN. The formalin/paraffin sections of liver samples were stained with a polyclonal antibody against GST-P. The percentages of GST-P (+) areas were determined from 10 fields using the Digital Camera System and Image-Pro Plus software. ***P < as compared to the control group. (c) Analysis of transforming growth factor-α (TGF-α) mRNA expression in the liver. Liver RNA was analyzed for TGF-α expression using real-time quantitative reverse transcriptase PCR (quantitative RT-PCR). TGF-α mRNA levels were normalized to those of β-actin mRNA. (d) Analysis of α-fetoprotein (AFP) mRNA expression in tumor nodules. Total RNA was prepared from 10 tumor nodules from group A or group B animals isolated at week 12 of DEN feeding, and analyzed for AFP expression by RT-PCR using two pairs of primers that amplify the 5′ and the 3′ coding regions of rat AFP, respectively. mRNA from normal, healthy liver was used as a control. “H2O” indicates that no RNA was included in the reaction. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Therapeutic effects of interferon-α (IFN-α) on hepatocellular carcinoma (HCC). Diethylnitrosamine solution was administered to the animals for 9 weeks in accordance with the protocol described in Materials and Methods, and they were given regular water for another 2 weeks and observed, so as to allow sufficient time for tumor progression. The animals were injected with phosphate-buffered saline (PBS) (n = 8), 1 × 109 plaque-forming units (PFU) of Ad/LacZ (n = 8), or 1 × 109 PFU of Ad/IFN-α (n = 11) through the portal vein. Two weeks later, the animals were killed, and the livers were removed for analysis. Representative liver pictures of the animals treated with (a) PBS, (b) Ad/LacZ, and (c) Ad/IFN-α are shown. Arrows indicate multifocal HCC nodules. (d) Tumor burden. Total tumor volumes were measured as described in the legend to Figure 3a. ***P < 0.001, analysis of variance (ANOVA). (e) glutathione S-transferase, placental form positive [GST-P (+)] area. The percentages of GST-P (+) areas were determined from 10 different fields using the Digital Camera System and Image Pro Plus software. **P < 0.005, ANOVA. Representative pictures of GST-P (+) areas in the livers of the animals treated with (f) PBS, (g) Ad/LacZ, and (h) Ad/IFN-α are shown. The images are shown at ×50 original magnification.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Amelioration of liver cirrhosis by interferon-α (IFN-α) treatment. (a) Differences in hydroxyproline levels. Before administering the adenovirus injection, the animals were partially hepatectomized in order to obtain pre-treatment liver samples. After therapy, liver hydroxyproline levels were measured from pre-treatment and post-treatment livers. The values represent the differences ±SEM of hydroxyproline levels between the post-treatment liver and the pre-treatment liver. (b) Sirius red staining. The percentages of Sirius red (+) areas in the post-treatment liver samples were determined from 10 different fields using the Digital Camera System and Image-Pro Plus software. ***P < 0.001, analysis of variance. The images are shown at ×50 original magnification. PBS, phosphate-buffered saline.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Mechanisms associated with the antitumor activity of IFN-α gene therapy. The OCT cryosections of the tumor regions from the animals treated with phosphate-buffered saline (PBS), Ad/LacZ, or Ad/IFN-α were examined for (a) tumor-infiltrating CD4+ and CD8+ lymphocytes, using mouse anti-rat CD4 and CD8 antibodies, respectively. (b) Proliferative cells were visualized using a mouse anti-proliferating cell nuclear antigen (anti-PCNA) antibody. (c) Apoptotic cells were visualized using a TUNEL in situ detection kit. (d) Microvessel density was visualized using a mouse anti-CD31 antibody. CD4+ and CD8+ cells in a were counted from 50 randomly chosen fields, whereas the positive cells in b–d were counted from 10 fields. The data represent the mean value ± SEM. ***P < 0.001, analysis of variance. (e) Transforming growth factor-α (TGF-α) mRNA expression. The animals were partially hepatectomized before adenovirus treatment in order to obtain pre-treatment liver samples. RNA was isolated from pre-treatment and post-treatment livers, and TGF-α mRNA expression was analyzed using real-time qRT-PCR. TGF-α mRNA levels were normalized to β-actin mRNA levels. The data represent the change in mRNA levels in the post-treatment liver expressed as multiples of those in the pre-treatment liver. Three animals from each treatment group were analyzed. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Transforming growth factor-β (TGF-β) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA levels after interferon-α (IFN-α) treatment. As described in the legend to Figure 6e, RNA was isolated from pre-treatment and post-treatment livers, and analyzed for (a) TGF-β and (b) TIMP-1 mRNA expression using real-time quantitative reverse transcriptase PCR. The expression levels were normalized to β-actin mRNA levels. The data represent the change in mRNA levels in the post-treatment liver expressed as multiples of those in the pre-treatment liver. Three animals from each treatment group were analyzed. **P < 0.005, analysis of variance. PBS, phosphate-buffered saline.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions