Biomedical subjects

Stefan Kubicka

Publications and source records attributed to Stefan Kubicka.

27 records · Page 2Linked to original sources

PubMed2003

Caspase 8 small interfering RNA prevents acute liver failure in mice.

A major concern in therapy of acute liver failure is protection of hepatocytes to prevent apoptosis and maintain liver function. Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells. To evaluate the therapeutic efficacy of siRNA in vivo we used different mouse models of acute liver failure. We directed 21-nt siRNAs against caspase 8, which is a key enzyme in death receptor-mediated apoptosis. Systemic application of caspase 8 siRNA results in inhibition of caspase 8 gene expression in the liver, thereby preventing Fas (CD95)-mediated apoptosis. Protection of hepatocytes by caspase 8 siRNA significantly attenuated acute liver damage induced by agonistic Fas (CD95) antibody (Jo2) or by adenovirus expressing Fas ligand (AdFasL). However, in a clinical situation the siRNAs most likely would be applied after the onset of acute liver failure. Therefore we injected caspase 8 siRNA at a time point during AdFasL- and adenovirus wild type (Adwt)-mediated liver failure with already elevated liver transaminases. Improvement of survival due to RNA interference was significant even when caspase 8 siRNA was applied during ongoing acute liver failure. In addition, it is of particular interest that caspase 8 siRNA treatment was successful not only in acute liver failure mediated by specific Fas agonistic agents (Jo2 and AdFasL) but also in acute liver failure mediated by Adwt, which is an animal model reflecting multiple molecular mechanisms involved in human acute viral hepatitis. Consequently, our data raise hope for future successful application of siRNA in patients with acute liver failure.

Acute Disease↗

PubMed2003

A telomerase-dependent conditionally replicating adenovirus for selective treatment of cancer.

The catalytic component of human telomerase reverse transcriptase (hTERT) is not expressed in most primary somatic human cells, whereas the majority of cancer cells reactivate telomerase by transcriptional up-regulation of hTERT. Several studies demonstrated that the hTERT promoter can be used to restrict gene expression of E1-deleted replication defective adenoviral vectors to telomerase-positive cancer cells. In this study, a conditionally replicating adenovirus (hTERT-Ad) expressing E1A genes under control of a 255-bp hTERT-promoter was constructed. Additionally, an internal ribosomal entry site-enhanced green fluorescent protein cassette was inserted downstream of the E1B locus to monitor viral replication in vivo. Adenoviral replication of hTERT-Ad and enhancement of enhanced green fluorescent protein expression could be observed in all investigated telomerase-positive tumor cell lines. In contrast, hTERT-Ad infection of telomerase-negative primary human hepatocytes did not result in significant replication. The capability of hTERT-Ad to induce cytopathic effects in tumor cells was comparable with that of adenovirus wild type and significantly higher compared with ONYX-015, regardless of the p53 status of the tumor cells. Single application of low-dose hTERT-Ad to tumor xenografts led to significant inhibition of tumor growth, confirming the potential therapeutic value of conditionally replicative adenoviral vectors. These in vivo experiments also revealed that hTERT-Ad-mediated oncolysis was more efficient than ONYX-015 treatment. These results demonstrate that expression of E1A under transcriptional control of the hTERT promoter is sufficient for effective telomerase-dependent adenovirus replication as a promising perspective for the treatment of the majority of epithelial tumors.

Adenoviridae↗

PubMed2003

Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA.

BACKGROUND & AIMS: Hepatitis B virus (HBV) causes acute and chronic infections that may result in severe liver diseases. Animal models to study new treatment options in vivo have several drawbacks. Therefore, we were interested to establish a new small animal model in which HBV replication and especially new treatment options can be studied easily. METHODS: Naked DNA of an HBV replication competent vector was transferred via tail vein into NMRI mice. HBV replication was studied in serum and liver of the animals. HBV replication was modulated by treatment through siRNA and nucleoside analogues. RESULTS: Tail vein transfer of a HBV replication competent construct resulted in expression of HBV-specific transcripts in the liver, and up to 10% of hepatocytes became HBc- and HBsAg-positive. HBeAg, HBsAg, and viral DNA could be detected in the serum of the animals, followed by the induction of HBV-specific cellular immune responses. Nucleoside treatment of the mice resulted in reduced polymerase activity in the liver. Additionally, siRNA transfer in the animals led to a significant reduction of HBsAg and/or eventually HBeAg expression, which was dependent on the localization of the complementary sequence in the HBV genome. CONCLUSIONS: We have established a mouse model to study HBV replication and to investigate new and existing treatment approaches in vivo. Interestingly, siRNA seems a promising innovative treatment option to inhibit specifically HBV replication in vivo.

Adenine↗

PubMed2003

Interleukin 6/gp130-dependent pathways are protective during chronic liver diseases.

The contribution of the acute phase inducer interleukin 6 (IL-6) in the pathogenesis of liver diseases is yet unclear. Our analysis showed enhanced expression of IL-6 in livers derived from patients with acute and chronic liver diseases. Additionally, IL-6 plasma levels were significantly increased in patients with chronic liver diseases and showed an inverse correlation with biochemical markers of liver function and a positive correlation with inflammatory markers, signs of portal hypertension, and the degree of liver fibrosis. To prove the relevance of these clinical findings, we applied the tetrachlorcarbonide (CCl(4)) model to conditional knockout animals (Cre/loxP system) for gp130, the common signal transducer of IL-6 family cytokines. Cre recombinases were expressed through a hepatocyte (AlfpCre) and a ubiquitous (MxCre) control element. Gp130 deleted mice had a totally abolished STAT3 activation and acute phase response induction, but gp130 deletion had no effect on the degree of acute liver injury and subsequent hepatocyte proliferation. In contrast, during chronic liver injury induced by biweekly application of CCl(4), deletion of the gp130 receptor in nonparenchymal liver cells and not hepatocytes resulted in fibrosis progression. In conclusion, our experiments indicate an involvement of IL-6 in the pathogenesis of liver diseases and suggest a protective role of IL-6/gp130-dependent pathways in nonparenchymal liver cells during fibrosis progression in chronic liver diseases. (Hepatology 2003;38:218-229).

Acute Disease↗

PubMed2002

Involvement of TRAIL and its receptors in viral hepatitis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill a broad spectrum of tumor cells but appears to be nontoxic to most normal cells. Because there are conflicting data about the hepatotoxicity of TRAIL, we investigated the physiological function of TRAIL and its receptors in the liver. Hepatocytes are sensitive for FasL- and TRAIL-mediated apoptosis in vitro, but TRAIL induces no apoptosis in healthy livers in vivo. Using mouse models of adenoviral hepatitis and livers of patients with hepatitis infection, we could demonstrate that apoptosis in virally infected hepatocytes is mediated by TRAIL receptor DR5 and TRAIL. In contrast to FasL, TRAIL-mediated apoptosis of hepatocytes in vivo is triggered through viral infection. The TRAIL receptor/ligand system enables the organisms to specifically kill virus-infected hepatocytes, whereas normal uninfected hepatocytes in vivo are resistant to TRAIL-mediated apoptosis. Overexpression of TRAIL in the liver after viral infection is not dependent on lymphocytes, natural killer, or Kupffer cells, which indicates that the TRAIL receptor/ligand system is a paracrine mechanism of hepatocytes against virally infected cells. Our results suggest that TRAIL might be used not only for cancer therapy but also for therapy of patients with viral hepatitis to selectively eliminate infected hepatocytes and limit viral replication.

Adenoviridae↗

PubMed2002

p53 mutation pattern and expression of c-erbB2 and c-met in gastric cancer: relation to histological subtypes, Helicobacter pylori infection, and prognosis.

The molecular mechanisms of Helicobacter pylori associated tumor development are poorly understood. The spectra of genetic alterations in neoplasms may provide clues to the molecular carcinogenesis of a tumor and may be relevant for the prognosis of the patients. We investigated the p53 mutation pattern and the protein expression of p53, c-erbB2, and c-met in 42 gastric cancers and correlated these alterations with H. pylori infection, histological subtypes and survival of the patients after curative resection. There were no differences in the incidences of the expression of p53, c-erbB2, and c-met in the tumor tissues according to H. pylori infection. Fifteen p53 mutations in 12 (29%) tumors were identified. More p53 mutations were found in patients with positive serology for H. pylori (43% vs 14%). This difference was not significant, but the small sample size may be insufficient to detect a potential statistical difference. There was neither a H. pylori-associated p53 hot-spot codon mutation nor a H. pylori characteristic mutational pattern of p53. Positive lymph nodes (P = 0.0061) and p53 mutations (P = 0.0035) were the only significant bad prognostic markers for survival after curative resection of the gastric cancers in our study. Our study does not indicate a unique molecular mechanism of p53 mutagenesis through H. pylori infection. The fact that p53 mutations were significantly correlated with poor survival of patients after potentially curative resection of gastric cancer may have clinical implications for multimodal therapies.

Genes, erbB-2↗

PubMed2002

VP22-mediated intercellular transport of p53 in hepatoma cells in vitro and in vivo.

The capacity of VP22 chimeric proteins to spread from the primary transduced cell to surrounding cells could improve gene therapy approaches, especially in cancer therapy. However, there are conflicting data about VP22-mediated intercellular trafficking in different studies. To assess the role of VP22 in gene therapy of hepatocellular carcinomas (HCCs) we constructed expression vectors for N- and C-terminal versions of VP22-p53 fusion proteins and investigated the VP22-mediated shuttle effect in hepatoma cells by cotransfection experiments. VP22-mediated trafficking was not detectable in hepatoma cells in vitro by fluorescence microscopy, but reporter gene transactivation assays demonstrated intercellular trafficking of functional VP22-p53 in vitro. For in vivo experiments, the recombinant adenoviruses Ad5CMVp53 and Ad5CMVp53-VP22 were constructed. In contrast to the in vitro experiments intercellular trafficking of VP22-p53 could be observed in subcutaneous tumors of hepatoma cells by fluorescence microscopy, indicating a stronger shuttle effect in solid tumors compared to cell culture experiments. Because spread of p53-VP22 in liver tumors was correlated with enhanced apoptosis of hepatoma cells VP22-mediated trafficking of potential therapeutic proteins may improve the results of gene therapy of HCCs.

Adenoviridae↗

PubMed2002

Gene therapy by intrahepatic and intratumoral trafficking of p53-VP22 induces regression of liver tumors.

BACKGROUND & AIMS: VP22-mediated intercellular transport provides an approach to deliver functional chimeric proteins into a high percentage of target cells. The aim of this study was to evaluate the efficacy of p53/VP22 fusion protein in gene therapy of liver tumors. METHODS: Expression vectors of N- and C-terminal fusion proteins of p53 and VP22 were subcloned and transcriptional properties of chimeric proteins were assessed by luciferase assays. Adenoviral vectors expressing p53 wild type (AdGFP/p53wt) and p53-VP22 (AdGFP/p53-VP22) were generated to investigate the VP22-mediated spreading in normal liver and in liver tumors in vivo by green fluorescent protein fluorescence and p53 immunohistochemistry. Gene therapy was investigated in subcutaneous and preclinical orthotopic animal tumor models after subcutaneous and intra-arterial administration of the adenoviruses, and tumor growth was assessed by direct calibration and magnetic resonance imaging. RESULTS: p53-VP22 showed enhanced transcriptional activity compared with p53 wild type. VP22-mediated intercellular transport of p53 could be observed in the normal liver and in liver tumors in vivo and was correlated with increased antitumor efficacy of gene therapy and improved survival of the animals. CONCLUSIONS: Fusion of VP22 to p53 strongly improves the results of p53 replacement gene therapy. Furthermore, the demonstrated VP22-mediated intercellular transport in the liver could be important for other strategies in liver gene therapy, providing a tool for enhancing the effect of gene therapy in liver diseases such as metabolic disorders or viral hepatitis.

Adenoviridae↗

PubMed2002

Jun kinase modulates tumor necrosis factor-dependent apoptosis in liver cells.

Tumor necrosis factor (TNF) triggers distinct pathways in liver cells through TNF receptor 1 (TNF-R1) via adapter molecules, including the intracellular cascades leading to apoptosis, nuclear factor-kappa B (NF-kappa B), and Jun kinase (JNK) activation. TNF-dependent activation of NF-kappa B induces the transcription of antiapoptotic genes that renders liver cells resistant against TNF-induced apoptosis. In contrast, the role of JNK during TNF-induced apoptosis is less clear, so we studied its role during this process. Hepatoma cells treated with TNF and cycloheximide undergo apoptosis, which is proceeded by a strong activation of JNK. Adenoviral vectors (adv) were tested to block TNF-dependent JNK activation selectively. An adv expressing dominant-negative (dn) TRAF2 inhibited only JNK and not ERK or NF-kappa B activation. However, the effect of inhibiting JNK activation with a dn TAK1 virus was also specific but was stronger than that via dn TRAF2. In further experiments, the inhibitory effect of dn TAK1 on JNK was used to define its role during TNF-dependent apoptosis. Inhibition of JNK by adv dn TAK1 resulted in an earlier and stronger induction of apoptosis. Interestingly, TAM67, a dn form of c-Jun, did not mediate the JNK-dependent effect on TNF-dependent apoptosis, indicating that other molecular targets are essential to confer this mechanism. However, the modified apoptosis pattern could be inhibited by adv expressing Bcl-2 or dn FADD. In conclusion, we define TAK1 as a kinase specifically involved in TNF-induced JNK activation in hepatoma cells and show that JNK transduces antiapoptotic signals, which modulate the strength and time course of FADD-dependent cell death involving mitochondrial permeability transfer.

Adaptor Proteins, Signal Transducing↗