Biomedical subjects

Ulrich M Lauer

Publications and source records attributed to Ulrich M Lauer.

17 recordsLinked to original sources

PubMed2006

Optimizing vector application for gene transfer into human hepatoblastoma cells.

Gene targeting is currently of distinct interest as an innovative additive treatment option in various malignancies. Its role in pediatric liver tumors has not yet been evaluated thoroughly. For the first time the authors systematically analyzed both lipid-based transfection as well as transduction with adenovirus vectors (Ad) and Sendai virus vectors (SeVV) in order to optimize gene transfer into hepatoblastoma (HB) cells. Two HB cell lines were infected with Ad or SeVV coding for green fluorescent protein (Ad-GFP, SeVV-GFP); transduction efficiencies and apoptosis were assessed using flow cytometry. Furthermore, lipofection of HB cell lines with plasmid-constructs comprising liver-specific promoters was performed using Lipofectamine 2000 and FuGENE 6; lipofection efficiency was monitored by flow cytometry, microscopy, and luciferase activity. The Ad-GFP showed higher transduction rates (61-86%) than the SeVV-GFP (4-24%) depending on the HB cell line used. Infections with first generation SeVV vectors (SeVV-GFP) led to increased target cell apoptosis (7-43%) compared to Ad-GFP (4-16%). The Lipofectamine 2000 revealed a higher transfection efficiency than the FuGENE 6 for both HB cell lines tested. The liver-specific promoters were found to be differently active in the HB cell lines. This study delineates recombinant adenovirus vectors as a promising tool for gene transduction in the HB cells. Furthermore, enhanced activity of the liver-specific promoters in HUH6 cells compared to HepT1 cells supports the observation of varying biological behavior in histologically differing HB tissues.

Adenoviridae↗

PubMed2006

HDAC inhibitor treatment of hepatoma cells induces both TRAIL-independent apoptosis and restoration of sensitivity to TRAIL.

Hepatocellular carcinoma (HCC) displays a striking resistance to chemotherapeutic drugs or innovative tumor cell apoptosis-inducing agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Recently, we found 2 histone deacetylase inhibitors (HDAC-I), valproic acid and ITF2357, exhibiting inherent therapeutic activity against HCC. In TRAIL-sensitive cancer cells, the mechanism of HDAC-I-induced cell death has been identified to be TRAIL-dependent by inducing apoptosis in an autocrine fashion. In contrast, in HCC-derived cells, a prototype of TRAIL-resistant tumor cells, we found a HDAC-I-mediated apoptosis that works independently of TRAIL and upregulation of death receptors or their cognate ligands. Interestingly, TRAIL resistance could be overcome by a combinatorial application of HDAC-I and TRAIL, increasing the fraction of apoptotic cells two- to threefold compared with HDAC-I treatment alone, whereas any premature HDAC-I withdrawal rapidly restored TRAIL resistance. Furthermore, a tumor cell-specific downregulation of the FLICE inhibitory protein (FLIP) was observed, constituting a new mechanism of TRAIL sensitivity restoration by HDAC-I. In contrast, FLIP levels in primary human hepatocytes (PHH) from different donors were upregulated by HDAC-I. Importantly, combination HDAC-I/TRAIL treatment did not induce any cytotoxicity in nonmalignant PHH. In conclusion, HDAC-I compounds, exhibiting a favorable in vivo profile and inherent activity against HCC cells, are able to selectively overcome the resistance of HCC cells toward TRAIL. Specific upregulation of intracellular FLIP protein levels in nonmalignant hepatocytes could enhance the therapeutic window for clinical applications of TRAIL, opening up a highly specific new treatment option for advanced HCC.

Antineoplastic Agents↗

PubMed2006

In vitro gene targeting in human hepatoblastoma.

Poor treatment results in advanced hepatoblastoma (HB) made alternative treatment approaches desirable. Gene-directed tumor therapy is increasingly investigated in different malignancies. The aim of this study was to analyze possible alternatives of gene transfer into HB cells and to study therapeutic applications based on different strategies. Liposomal transfection of HB cells was assessed using liver-specific promoters, and adenovirus and Sendai virus transductions were performed in vitro. Transfer efficiencies were measured via flow cytometry determining expression of vector-encoded marker gene green fluorescent protein. Gene silencing of the anti-apoptotic bcl-2 gene in HUH6 cells was performed using lipofection of small interfering RNA (siRNA). Additionally, suicide gene therapy was carried out through a yeast-derived cytosine deaminase (YCD)-combined yeast uracil phosphoribosyltransferase (YUPRT)-based adenovirus-mediated gene transfer, leading to a potent intracellular prodrug transformation of 5-fluorocytosine into 5-fluorouracil. Treatment efficiencies were monitored via MTT viability assay. Highest gene transfer rates (86%) were observed using adenovirus transduction. We furthermore observed a significant therapeutic effect of adenovirus-mediated YCD::YUPRT suicide gene transfer. Liposomal-mediated anti-bcl-2 siRNA transfer led to a significant improvement of cisplatin treatment in HUH6 cells. Liver-specific promoters were found to be strongly active in HUH6 cells (mixed HB-derived), but less active in HepT1 cells (embryonal HB-derived). Liposomal transfection and viral transduction are effective approaches to genetically manipulate HB cells in vitro. For the first time, we demonstrate a positive effect of siRNA gene silencing in this malignancy. Additionally, we successfully investigated a model of adenovirus-based suicide gene therapy in HB cell cultures. Our data strongly encourage further studies assessing these alternative treatment approaches.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Bifunctional chimeric SuperCD suicide gene -YCD: YUPRT fusion is highly effective in a rat hepatoma model.

AIM: To investigate the effects of catalytically superior gene-directed enzyme prodrug therapy systems on a rat hepatoma model. METHODS: To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine (5-FC), we generated a chimeric bifunctional SuperCD suicide gene, a fusion of the yeast cytosine deaminase (YCD) and the yeast uracil phosphoribosyltransferase (YUPRT) gene. RESULTS: In vitro stably transduced Morris rat hepatoma cells (MH) expressing the bifunctional SuperCD suicide gene (MH SuperCD) showed a clearly marked enhancement in cell killing when incubated with 5-FC as compared with MH cells stably expressing YCD solely (MH YCD) or the cytosine deaminase gene of bacterial origin (MH BCD), respectively. In vivo, MH SuperCD tumors implanted both subcutaneously as well as orthotopically into the livers of syngeneic ACI rats demonstrated significant tumor regressions (P<0.01) under both high dose as well as low dose systemic 5-FC application, whereas MH tumors without transgene expression (MH naive) showed rapid progression. For the first time, an order of in vivo suicide gene effectiveness (SuperCD>> YCD>>BCD>>>negative control) was defined as a result of a direct in vivo comparison of all three suicide genes. CONCLUSION: Bifunctional SuperCD suicide gene expression is highly effective in a rat hepatoma model, thereby significantly improving both the therapeutic index and the efficacy of hepatocellular carcinoma killing by fluorocytosine.

Animals↗

PubMed2005

Severe impairment of dendritic cell allostimulatory activity by Sendai virus vectors is overcome by matrix protein gene deletion.

Delivery of Ags to dendritic cells (DCs) plays a pivotal role in the induction of efficient immune responses ranging from immunity to tolerance. The observation that certain viral pathogens are able to infect DCs has led to a concept in which applications of recombinant viruses are used for Ag delivery with the potential benefit of inducing potent Ag-specific T cell responses directed against multiple epitopes. As a prerequisite for such an application, the infection of DCs by recombinant viruses should not interfere with their stimulatory capacity. In this context, we could show that an emerging negative-strand RNA viral vector system based on the Sendai virus (SeV) is able to efficiently infect monocyte-derived human DCs (moDCs). However, after infection with SeV wild type, both the response of DCs to bacterial LPS as a powerful mediator of DC maturation and the allostimulatory activity were severely impaired. Interestingly, using various recombinant SeV vectors that were devoid of single viral genes, we were able to identify the SeV matrix (M) protein as a key component in moDC functional impairment after viral infection. Consequently, use of M-deficient SeV vectors preserved the allostimulatory activity in infected moDCs despite an efficient expression of all other virally encoded genes, thereby identifying M-deficient vectors as a highly potent tool for the genetic manipulation of DCs.

Apoptosis↗

PubMed2005

Natural killer cell-mediated lysis of hepatoma cells via specific induction of NKG2D ligands by the histone deacetylase inhibitor sodium valproate.

Natural killer (NK) cells as components of the innate immunity substantially contribute to antitumor immune responses. However, the tumor-associated ligands engaging activating NK cell receptors are largely unknown. An exception are the MHC class I chain-related molecules MICA and MICB and the UL16-binding proteins (ULBP) which bind to the activating immunoreceptor NKG2D expressed on cytotoxic lymphocytes. A therapeutic induction of NKG2D ligands that primes cancer cells for NK cell lysis has not yet been achieved. By microarray studies, we found evidence that treatment of human hepatocellular carcinoma cells with the histone deacetylase inhibitor (HDAC-I) sodium valproate (VPA) mediates recognition of cancer cells by cytotoxic lymphocytes via NKG2D. VPA induced transcription of MICA and MICB in hepatocellular carcinoma cells, leading to increased cell surface, soluble and total MIC protein expression. No significant changes in the expression of the NKG2D ligands ULBP1-3 were observed. The induction of MIC molecules increased lysis of hepatocellular carcinoma cells by NK cells which was abolished by addition of a blocking NKG2D antibody. Importantly, in primary human hepatocytes, VPA treatment did not induce MIC protein expression. Taken together, our data show that the HDAC-I VPA mediates specific priming of malignant cells for innate immune effector mechanisms. These results suggest the clinical evaluation of HDAC-I in solid tumors such as hepatocellular carcinoma, especially in combination with immunotherapy approaches employing adoptive NK cell transfer.

Carcinoma, Hepatocellular↗

PubMed2005

Combined endostatin/sFlt-1 antiangiogenic gene therapy is highly effective in a rat model of HCC.

Hepatocellular carcinoma (HCC) is regarded as a suitable target for antiangiogenic strategies. However, antiangiogenic agents aimed at single targets can be neutralized by upregulation of other proangiogenic factors. Therefore, combined approaches addressing at least two angiogenic targets should be more effective. Employing an appropriate rat hepatoma model, we examined the effects of sFlt-1 (soluble vascular endothelial growth factor [VEGF] receptor 1 as an indirect inhibitor of angiogenesis) and endostatin (a direct inhibitor of angiogenesis) in both single-agent as well as combined approaches under in vitro and in vivo conditions. Similar to human HCC, rat Morris hepatoma (MH) cells secreted high levels of VEGF, but no endogenous sFlt-1. Parental MH or MHES(r) cells, stably expressing rat endostatin, were adenovirally transduced either with AdsFlt-1 (encoding sFlt-1) or control vector Adnull (containing no transgene), followed by subcutaneous inoculation into syngeneic ACI rats. Compared with MH/Adnull cells, expressing no antiangiogenic factors at all, tumor weights were reduced fourfold in the MHES(r)/Adnull group, 19-fold in the MH/AdsFlt-1-group, and 77-fold in the MHES(r)/AdsFlt-1 combination therapy group. Analysis of variance did not show a significant interaction between the effects of the two factors ES(r) and sFlt-1; their effects multiplied. In conclusion, combined expression of sFlt-1 and endostatin effectively suppresses HCC growth under in vivo conditions. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Angiogenesis Inhibitors↗

PubMed2005

Apoptosis on hepatoma cells but not on primary hepatocytes by histone deacetylase inhibitors valproate and ITF2357.

BACKGROUND/AIMS: Due to a particular resistance against conventional chemotherapeutics, palliative treatment of hepatocellular carcinomas (HCC) is highly ineffective. Recent demonstration of both proliferation-inhibition and apoptosis of hepatoma cells by a histone deacetylase inhibitor (HDAC-I) treatment opens up a promising new approach. However, little is known about tumor cell death mechanisms and HDAC-I influences on healthy hepatocytes. METHODS: HDAC-I substances with favourable in vivo profiles, valproate (VPA) and ITF2357, were investigated on HCC cell lines and primary human hepatocytes (PHH). Histone acetylation and apoptosis-modulating proteins were investigated by western-blotting, proliferation by sulforhodamin B binding, toxicity by enzyme release, apoptosis by FACS analysis. RESULTS: VPA and ITF2357 inhibited proliferation in HCC cell lines. Both substances induced considerable cellular damage in HCC-derived cells, but PHH tolerated these substances well. A downregulation of anti- and upregulation of proapoptotic factors was found. Moreover, Bcl-X(L) transfection into HCC cells abrogated apoptosis induced by both substances, indicating that modulation of intracellular pro- and anti-apoptotic proteins is a key event in VPA or ITF2357 induced tumor-cell death. CONCLUSIONS: Preferential induction of cell death in HCC-derived cell lines, without toxicity in PHH, demonstrates the potential of VPA and ITF2357 to become promising new tools in the fight against HCC.

Apoptosis↗

PubMed2005

Protein transduction with bacterial cytosine deaminase fused to the TLM intercellular transport motif induces profound chemosensitivity to 5-fluorocytosine in human hepatoma cells.

BACKGROUND/AIMS: This study investigates the application of protein based therapeutic suicide enzyme/prodrug approaches providing novel means for both safe and effective local therapeutic regimes in solid tumors. METHODS: Employing a novel cell permeable peptide, known as the translocation motif (TLM) of hepatitis B virus, E. coli cytosine deaminase (BCDase) suicide fusion proteins were generated. RESULTS: TLM fusion proteins formed hexamers (as do parental wtBCDase proteins) and retained the specific enzymatic activity of cytosine conversion to uracil also being comparable to parental wtBCDase proteins. However, only BCDase-TLM fusion proteins, but not TLM-BCDase fusion nor parental wtBCDase proteins were found to be taken up to the cytoplasm of target cells as demonstrated both by confocal laser scanning microscopy and cell fractionation. Uptake of BCDase-TLM worked both efficiently and rapidly and was found to be independent from the endosomal pathway. Since BCDase-TLM fusion proteins completely retained their suicide enzymatic activity in the course of translocation across the plasma membrane their usage as profound inducers of chemo-sensitivity to 5-FC strongly is suggested. CONCLUSIONS: Future therapeutic local application of cell-permeable BCDase-TLM fusion proteins together with a systemic 5-FC prodrug application could result in profound antitumor activities without apparent side effects.

Animals↗

PubMed2004

Efficient propagation of single gene deleted recombinant Sendai virus vectors.

Recombinant Sendai virus vectors (SeVV) have become an attractive tool for basic virological as well as for gene transfer studies. However, to (i) reduce the cellular injury induced by basic recombinant SeV vectors (encoding all six SeV genes as being present in SeV wild-type (wt) genomes) and to (ii) improve SeV vector safety, deletions of viral genes are necessary for the construction of superior SeVV generations. As a strong expression system recombinant replication-incompetent adenoviruses, coding for SeV proteins hemagglutinin-neuraminidase (HN), fusion (F), or matrix (M), were generated and successfully employed for the propagation of single gene deleted (DeltaHN, DeltaF, DeltaM) recombinant SeVV. Further investigations of the propagation procedures required for single gene deleted recombinant SeVV demonstrated (i) modifications of the cell culture medium composition as well as (ii) incubation with vitamin E as crucial steps for the enhancement of SeVV-DeltaHN, -DeltaF, or -DeltaM viral particle yield. Such optimized propagation procedures even led to a successful propagation of HN-deleted viral particles (SeVV-DeltaHN), which has not been reported before.

Adenoviridae↗

PubMed2004

Recombinant Sendai virus for efficient gene transfer to human airway epithelium.

Recombinant Sendai virus (rSeV) infects respiratory epithelial cells in animal models and cultures of undifferentiated human nasal cells. It was the aim of this study to investigate the capability of rSeV to express a transgene in human airway epithelium. Differentiated human airway epithelial cells were generated using air-liquid interface culture techniques. Application of rSeV coding for green fluorescence protein (GFP) onto the apical surface (using a multiplicity of infection of 3) resulted in expression of the transgene in more than 90% of the cells followed by decreasing numbers of positive cells during the observation time of 3 weeks. The infection of human respiratory epithelial cells is mediated by sialic acid residues at the apical surface. Despite the secretion of interleukin (IL)-8 and the replication of rSeV in the epithelial cells, the authors could not detect any cytopathic effect after the infection. In conclusion, rSeV infects differentiated human airway epithelial cells with high efficiency. Transgene expression is transient and accompanied by the secretion of an inflammatory cytokine.

Bronchi↗

PubMed2003

Adenoviral gene transfer of tumor necrosis factor-related apoptosis-inducing ligand overcomes an impaired response of hepatoma cells but causes severe apoptosis in primary human hepatocytes.

Ligands of the tumor necrosis factor family play key roles in liver pathogenesis. The ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is unique, because it is thought to be nontoxic to normal cells while killing a broad range of tumor cells. However, hepatocellular carcinoma is considered resistant to soluble TRAIL treatment. Therefore, a direct gene transfer of TRAIL to malignant cells is part of an alternative delivery strategy. We show that an adenoviral gene transfer (Ad-TRAIL) overcomes an impaired response of hepatocellular carcinoma cell lines to soluble TRAIL, but the transduction of primary human hepatocytes revealed a high number of apoptotic cells. Our data imply that Ad-TRAIL administration in vivo must either be restricted to tumor tissue or controlled by a tumor-specific promoter to avoid severe liver damage in human trials.

Adenoviruses, Human↗

PubMed2003

Sendai virus vectors as an emerging negative-strand RNA viral vector system.

The power to manipulate the genome of negative-strand RNA viruses, including the insertion of additional non-viral genes, has led to the development of a new class of viral vectors for gene transfer approaches. The murine parainfluenza virus type I, or Sendai virus (SeV), has emerged as a prototype virus of this vector group, being employed in numerous in vitro as well as animal studies over the last few years. Extraordinary features of SeV are the remarkably brief contact time that is necessary for cellular uptake, a strong but adjustable expression of foreign genes, efficient infection in the respiratory tract despite a mucus layer, transduction of target cells being independent of the cell cycle, and an exclusively cytoplasmic replication cycle without any risk of chromosomal integration. In this review we describe the current knowledge of Sendai virus vector (SeVV) development as well as the results of first-generation vector applications under both in vitro and in vivo conditions. So far, Sendai virus vectors have been identified to be a highly efficient transduction tool for a broad range of different tissues and applications. Future directions in vector design and development are discussed.

Animals↗

PubMed2003

Cell cycle independent infection and gene transfer by recombinant Sendai viruses.

A common problem for viral vectors in the field of somatic gene therapy is the dependence of an efficient cellular transduction on the cell cycle phase of target cells. An optimized viral vector system should therefore transduce cells in different cell cycle phases equally to improve transduction efficiencies. Recent observations that recombinant Sendai viruses (SeV) can infect a broad range of different tissues suggested SeV to be a good candidate for future gene therapeutic strategies in which dividing and non-dividing cells have to be reached. However, detailed data on the influence of distinct cell cycle phases on the infection of SeV or related viruses are missing. We report that synchronization of NIH 3T3 cells as well as contact inhibition of human fibroblast cells did not exhibit any negative influence on SeV infection rates. Furthermore, different attractive target tissues like human umbilical cord derived cells or primary human hepatocytes can be reached by SeV efficiently. As an important information for further cell cycle studies of paramyxoviruses we discovered surprisingly that the DNA polymerase inhibitor aphidicolin (induces a G(1)/M arrest) functions as an inhibitor of SeV but not of an adenoviral expression vector. In conclusion, the results demonstrate SeV based vector particles to be an ideal tool to reach equally cells coexisting in different cell cycle phases.

3T3 Cells↗

PubMed2003

Negative-strand RNA viral vectors: intravenous application of Sendai virus vectors for the systemic delivery of therapeutic genes.

Treatment by gene replacement is critical in the field of gene therapy. Suitable vectors for the delivery of therapeutic genes have to be generated and tested in preclinical settings. Recently, extraordinary features for a local gene delivery by Sendai virus vectors (SeVV) have been reported for different tissues. Here we show that direct intravenous application of SeVV in mice is not only feasible and safe, but it results in the secretion of therapeutic proteins to the circulation, for example, human clotting Factor IX (hFIX). In vitro characterization of first-generation SeVV demonstrated that secreted amounts of hFIX were at least comparable to published results for retroviral or adeno-associated viral vectors. Furthermore, as a consideration for application in humans, SeVV transduction led to efficient hFIX synthesis in primary human hepatocytes, and SeVV-encoded hFIX proteins could be shown to be functionally active in the human clotting cascade. In conclusion, our investigations demonstrate for the first time that intravenous administration of negative-strand RNA viral vectors may become a useful tool for the wide area of gene replacement requirements.

3T3 Cells↗

PubMed2003

Endothelial endostatin release is induced by general cell stress and modulated by the nitric oxide/cGMP pathway.

Endostatin is a 20 kDa carboxyl-terminal fragment of collagen XVIII that, when added exogenously, inhibits endothelial proliferation and migration in vitro and angiogenesis and tumor growth in vivo. Previous results showed endostatin/collagen XVIII labeling in few endothelial cells in human glioblastoma multiforme. We have now observed constitutive release of endostatin from one of four endothelial cell lines. Induction of endostatin release was observed after H2O2, an in vitro model of cell stress, CoCl2, a model of hypoxia, and by IFN-gamma challenge. Endostatin expression and release was reduced by the nitric oxide synthase inhibitors aminoguanidine and L-NAME and induced by the NO synthase-independent NO donors sodium nitroprusside (SNP) and spermine-NONO-ate. SNP-mediated endostatin induction was abrogated by the soluble guanylate cyclase inhibitor 1H-(1.2.4) oxadiazolo (4,3-A) quinoxalin-1-one. Adenoviral endostatin transduction resulted in the release of endostatin from endothelial cells and in down-regulation of iNOS (NOS2) and eNOS (NOS3), and surprisingly in a 10% induction of PCNA. These results describe the modulation of endostatin release by the NO signaling cascade and provide important new pharmacological information for the systemic induction of endogenous endostatin release by common NO donor pharmacotherapy.

Adult↗

PubMed2002

Caspase-8 and Apaf-1-independent caspase-9 activation in Sendai virus-infected cells.

Apoptotic cell death is of central importance in the pathogenesis of viral infections. Activation of a cascade of cysteine proteases, i.e. caspases, plays a key role in the effector phase of virus-induced apoptosis. However, little is known about pathways leading to the activation of initiator caspases in virus-infected host cells. Recently, we have shown that Sendai virus (SeV) infection triggers apoptotic cell death by activation of the effector caspase-3 and initiator caspase-8. We now investigated mechanisms leading to the activation of another initiator caspase, caspase-9. Unexpectedly we found that caspase-9 cleavage is not dependent on the presence of active caspases-3 or -8. Furthermore, the presence of caspase-9 in mouse embryonic fibroblast (MEF) cells was a prerequisite for Sendai virus-induced apoptotic cell death. Caspase-9 activation occurred without the release of cytochrome c from mitochondria and was not dependent on the presence of Apaf-1 or reactive oxygen intermediates. Our results therefore suggest an alternative mechanism for caspase-9 activation in virally infected cells beside the well characterized pathways via death receptors or mitochondrial cytochrome c release.

Animals↗