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Background/aims: To evaluate the long-term outcome of surgical and non-surgical local treatments of patients with hepatocellular carcinoma (HCC)., Methods: We stratified a cohort of 278 HCC patients using six independent predictors of survival according to the Vienna survival model for HCC (VISUM-HCC)., Results: Prior to therapy, 224 HCC patients presented with VISUM stage 1 (median survival 18 months) while 29 patients were classified as VISUM stage 2 (median survival 4 months) and 25 patients as VISUM stage 3 (median survival 3 months). A highly significant (p < 0.001) improved survival time was observed in VISUM stage 1 patients treated with liver resection (n = 52; median survival 37 months) or chemoembolization (TACE) and subsequent radiofrequency ablation (RFA) (n = 44; median survival 45 months) as compared to patients receiving chemoembolization alone (n = 107; median survival 13 months) or patients treated by tamoxifen only (n = 21; median survival 6 months). Chemoembolization alone significantly (p < or = 0.004) improved survival time in VISUM stage 1-2 patients but not (p = 0.341) in VISUM stage 3 patients in comparison to those treated by tamoxifen., Conclusion: Both liver resection or combined chemoembolization and RFA improve markedly the survival of patients with HCC., (2007 S. Karger AG, Basel)

Background/aims: Hepatocellular carcinomas (HCC) often show resistance to the effects of transforming growth factor-beta (TGF-beta). This study focuses on molecular mechanisms of this resistance to explore ways to overcome it., Methods: Transcription and protein expression of TGF-beta type I and type II receptors (TGF-betaRI/RII) were analyzed in clinical HCCs and the human hepatoma cell lines HuH-7 and HepG2. HuH-7 cells were transiently and stably transfected with a constitutively active TGF-betaRI mutant (CA TGF-betaRI). Resulting growth kinetics, integrin expression, invasiveness, TGF-beta-mediated activation of human plasminogen activator inhibitor type-1 (PAI-1) promoter and Smad expression were determined., Results: In clinical HCCs, there was less TGF-betaRII (6/10 cases) and more TGF-betaRI (8/10 cases) protein expression detectable in tumor compared to adjacent liver tissue. In HuH-7 cells, TGF-betaRII expression was likewise decreased. Cells transiently transfected with CA TGF-betaRI exhibited strong TGF-beta-related PAI-1 promoter activation. Stably transfected cells showed an attenuated response of the PAI-1 promoter, but increased Smad7 expression. Proliferation of stable clones was decreased. There was no change in integrin expression or invasiveness., Conclusions: Decreased TGF-betaRII protein expression might cause TGF-beta resistance in a subset of clinical HCCs. Stable transfection with CA TGF-betaRI reverses this in HuH-7 cells without increasing invasiveness., (Copyright 2005 S. Karger AG, Basel.)

Background/aims: Transgenic mice overexpressing hepatitis B x protein (HBx) show an increased susceptibility to mutations if exposed to mutagens. Also involved in HBx signalling, reactive oxygen intermediates (ROI) can induce DNA adducts such as 8-hydroxy-2'-deoxyguanosine that can in turn lead to G/T transversion mutations. Therefore, we investigated whether HBx expression increases the level of the mutational precursor 8-hydroxy-2'-deoxyguanosine in hepatocellular DNA., Methods: 8-hydroxy-2'-deoxyguanosine concentrations of DNA hydrolysates of HBx protein expressing HepG2 cells and livers of HBx transgenic mouse lines were determined electrochemically after HPLC fractionation., Results: 8-hydroxy-2'-deoxyguanosine concentrations in genomic DNA of HBx protein expressing cell lines correlated with the factor of transactivation. The 8-hydroxy-2'-deoxyguanosine levels were reduced after incubation of HBx recombinant cell lines with 0.1 or 1 mM of the antioxidant N-acetylcysteine. Hepatic 8-hydroxy-2'-deoxyguanosine concentrations in DNA of old transgenic mice were significantly, i.e. twofold, (p < 0.01) increased as compared to those of old nontransgenic or young transgenic controls and of control mice expressing a second HBV transactivator (MHBs(t76))., Conclusion: HBx expression results in elevated DNA adduct levels. This could reflect a direct inhibitory interaction of HBx with cellular repair mechanisms. Alternatively, this may be an effect of an increased generation of reactive oxygen intermediates through HBx., (Copyright 2004 S. Karger AG, Basel.)

Background and Aims: Adeno-associated virus (AAV) is the only known virus capable of site-specific genomic integration in human cells. Thus, AAV-based vectors may be an attractive option to achieve prolonged transgene expression in human cells. We therefore studied the minimal elements of gene therapy vectors necessary for stable integration and tested the effectiveness of this approach in hepatoma cells., Methods: Plasmids were constructed that contained a GFPneo fusion transgene with or without the AAV-inverted terminal repeats (ITRs). In addition, Rep protein was either encoded in CIS or supplied in TRANS by co-transfections. Stable clones were analyzed by Southern blotting for site-specific integration., Results: The ITRs alone conferred neither stable nor site-specific transgene integration. Expression of Rep protein in CIS or TRANS resulted in an increased frequency of integration regardless of the presence of ITRs. It was shown that in the absence of the ITRs, other Rep-binding site (RBS) like sequences such as the ColE1 sequence present in plasmid backbones can function as RBS. Site-specific integration was achieved in up to 26% of clones derived from hepatoma cells., Conclusion: Both expression of Rep proteins and inclusion of a RBS are necessary for enhanced and stable integration of AAV-based non-viral vectors. A novel two-plasmid system capable of achieving stable and site-specific gene transfer in hepatoma cells is introduced., (Copyright 2003 S. Karger AG, Basel)

Background/aims: Standard methods for hepatitis C virus (HCV) RNA quantification are time-consuming and often hampered by low sensitivity. Therefore, we aimed to test whether fluorescence correlation spectroscopy (FCS) could be used to read out HCV polymerase chain reactions (PCR)., Methods: A single-step reverse transcriptase (RT) PCR system was adjusted to the clinically relevant range of 1 x 10(3) to 5 x 10(6) HCV cDNA copies/ml serum. Unpurified amplification mixtures were analyzed by FCS and controlled by HPLC analysis., Results: The outcome of HCV RNA quantitation was nearly identical no matter whether FCS or HPLC techniques were used. FCS-generated standard curves displayed sufficient linearity to allow reproducible determinations. The intraserial variation of cDNA quantification after PCR amplification was +/-3.2%, the interserial variation +/-4.3%. Repeated quantifications of HCV genotype 1b RNA from the sera of 8 patients revealed titers from 1 x 10(4)-5 x 10(6) genome equivalents/ml. The results correlated significantly (r = 0.755; p = 0.03) with a widely used commercially available assay., Conclusion: FCS may become a useful tool for rapid and reproducible HCV RNA quantification in the future., (Copyright 2000 S. Karger AG, Basel)