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Sorin Armeanu

Publications and source records attributed to Sorin Armeanu.

18 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

Expression liver-directed genes by employing synthetic transcriptional control units.

AIM: To generate and characterize the synthetic transcriptional control units for transcriptional targeting of the liver, thereby compensating for the lack of specificity of currently available gene therapeutic vector systems. METHODS: Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting. RESULTS: A new bifunctionally-enhanced green fluorescent protein (EGFP)/neo(r) fusion gene cassette was generated, and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to "naturally" derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 (ApoB) promoter or the ornithin transcarbamoylase (OTC) enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor (HNF)1, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein (C/EBP) only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xApoB cassette construct, which fully complied with the data obtained by initial FACS analysis. CONCLUSION: Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver-directed expression of therapeutic genes.

Carcinoma, Hepatocellular↗
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

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↗
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↗
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↗
PubMed2002

Optimization of nonviral transfection: variables influencing liposome-mediated gene transfer in proliferating vs. quiescent cells in culture and in vivo using a porcine restenosis model.

Cationic liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ss-galactosidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling, low toxicity, and the lack of carcinogenicity or immunogenic reactions.

Animals↗
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↗
PubMed2002

Vascular endothelial growth factor (VEGF165) and its influence on angiogenesis versus arteriogenesis in different vascular beds.

PURPOSE: To use local gene delivery to determine any district-specific influence of vascular endothelial growth factor (VEGF(165)) on angiogenesis and arteriogenesis in arteries of distinct developmental origin. METHODS: Coronary and peripheral arteries were chronically occluded in 30 Pietrain pigs using a percutaneous approach and blinded stent-graft. DNA was delivered to the adventitia in dosages corresponding to 10% of the body weight-adapted amount used in clinical trials. The coronary arteries in 12 animals and the peripheral arteries in 12 animals were treated or used as controls (no occlusion or occlusion with transfection of the beta-galactosidase gene). Six additional animals were sacrificed at 1 or 3 weeks for expression analyses, while the other 24 animals were sacrificed at 5 months for expression analysis and histology. Angiography, polymerase chain reaction analyses, and immunohistochemistry were performed. RESULTS: Expression of the VEGF gene was observed at 1 and 3 weeks following application, while transfected DNA was detected up to 5 months. New collaterals formed around occluded coronary arteries (2.63 +/- 0.69 fold, p<0.05 versus 1.24 +/- 0.40 fold for peripheral arteries), and angiographic arterial area increase was more pronounced in coronary (2.49 +/- 0.59 fold, p<0.05) than peripheral arteries (1.49 +/- 0.05 fold). There was no collateralization surrounding occluded peripheral arteries, but new arterial branches were seen (2.0 +/- 0.28, p<0.05 versus 1.07 +/- 0.31 for coronary). CONCLUSIONS: The response to VEGF, whether it is predominantly angiogenesis or arteriogenesis, is dependent on the target vessel. These observed differences in the behavior of arteries may be related to their differing developmental origins, which may have important implications for future therapeutic strategies using VEGF in different vessels.

Animals↗
PubMed2000

Involvement of E-cadherin in the Development of Erythroid Cells; Subject Heading.

The cadherins represent a large family of structurally and functionally related cell adhesion molecules involved in morphogenesis of multicellular organisms and maintenance of solid tissues. In the hematopoietic system, however, almost nothing was known about the involvement of this family. PCR screening of RNA of human bone marrow mononuclear cells with specific primers for different classical cadherins revealed that members of this family are also expressed by bone marrow cells. Here we report that E-cadherin, which is mainly expressed by cells of epithelial origin, plays a critical role in the development of human erythrocytes. FACS analysis with human E-cadherin-specific antibodies and the use of immunoaffinity columns revealed that expression of E-cadherin is restricted to defined maturation stages of the erythropoietic cell lineage. Erythroblasts and normoblasts express E-cadherin, but mature erythrocytes do not. Lymphoid and all the other myeloid cell lineages do not express E-cadherin at any developmental stage. The differentiation of the erythroid lineage in vitro could be influenced by addition of anti-E-cadherin antibodies in a concentration dependent manner indicating a direct involvement of this cell adhesion molecule in the differentiation process. In line with these in vitro data is the finding that E-cadherin is down regulated during erythroleukemia on the developing erythroid cells. Our results suggest an unanticipated function of E-cadherin in the hematopoietic system.

Journal Article↗