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Michael Bitzer

Publications and source records attributed to Michael Bitzer.

At least 19 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

Synchronous gastrointestinal and musculoskeletal manifestations of different subtypes of inflammatory myofibroblastic tumor: CT, MRI and pathological features.

We describe a case of an unusual multicentric appearance of an inflammatory myofibroblastic tumor, consisting of multiple gastrointestinal masses with different growth patterns and simultaneous, distant, musculoskeletal manifestations. CT and MR imaging features demonstrated a different degree of lesion enhancement, which proved histologically to be related to an alternation of predominantly spindle cell areas with a myxoid-vascular IMT subtype. A clear separation of the imaging characteristics of this tumor's subtypes by correlation with the pathology was not possible because of the mixed histologic character of the tumor in all its locations. However, MRI signal in the T2-weighted sequence was lower in the spindle cell variant localized predominantly in the musculoskeletal system, while the gastrointestinal predominantly myxoid-vascular counterparts showed slightly higher signal in the T2-weighted sequence.

Aged↗
PubMed2004

Recurrent spinal fistula as result of a rare combination of a perimedullary and peridural spinal fistula: case report.

OBJECTIVES: We report on a patient with the combination of a peridural and a perimedullary spinal fistula, which manifested consecutively. The clinical course and diagnostic steps reveal important observations helpful in the management of this pathology. CASE PRESENTATION: A 61-year-old male patient presented with a six-month history of progressive weakness of the lower limbs. Magnetic resonance imaging revealed edema and dilated spinal veins of the lower thoracic spinal cord. Spinal angiography confirmed the diagnosis of spinal dural fistula at level T9 on the left. The patient underwent surgery and the fistula was surgically excised. Two months after initial improvement, the clinical symptoms of lower limb weakness recurred. On re-angiography a spinal perimedullary fistula was found at level T7 that was not apparent on the previous angiogram and on the postoperative control angiogram. The patient underwent surgery again, and the second fistula was also excised. The clinical symptoms subsequently improved. CONCLUSION: The interesting point in this case was the rare combination of a peridural and a perimedullary spinal fistula. They presented consecutively and could not be identified simultaneously on the first angiogram. Only after closure of the first fistula did the second become apparent. We believe that this may be a result of a postoperative pressure change in the venous system of the cord. After closure of the first fistula, the arterio-venous (AV) shunt of the second fistula developed gradually. The possibility of a second fistula should be considered in the presence of persistent edema of the cord on magnetic resonance imaging (MRI) and subsequent clinical deterioration.

Angiography↗
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↗
PubMed2004

The benefit of functional-anatomical imaging with [18F]fluorodeoxyglucose utilizing a dual-head coincidence gamma camera with an integrated X-ray transmission system in non-small cell lung cancer.

AIM: To evaluate functional-anatomical imaging with 2-[F]fluoro-2-deoxy-D-glucose (F-FDG) utilizing a dual-head coincidence gamma camera with an integrated X-ray transmission system for attenuation correction, anatomical mapping, and image fusion compared to conventional diagnostics by computed tomography (CT) in non-small cell lung cancer (NSCLC). METHODS: Thirty-five patients with NSCLC underwent FDG imaging of the thoracic area using a dual-head coincidence gamma camera (DHC) with an integrated X-ray transmission system. State-of-the-art CT scans had been performed before. Whole-body dedicated FDG positron emission tomography (PET) was performed immediately prior to DHC. Staging by CT and DHC, and DHC with integrated image fusion (FDHC) were re-evaluated with regard to detectable lesions, correct anatomical diagnoses, and clinical impact. Results of DHC and PET were compared for analysis of limitations of DHC. RESULTS: One hundred and thirteen tumour lesions were identified by CT. DHC detected 128 lesions overall: 102 true positive CT lesions were confirmed, 25 additional lesions were detected which affected staging in eight patients, and one false positive lung lesion did not show up in DHC. Nine CT lesions were missed by DHC (lymph node and lung). PET detected 150 areas of focally enhanced uptake, delivering two false positive results (nuchal muscles, pneumonia). Final evaluation confirmed 148 malignant lesions. Compared to CT, the results of DHC changed staging or treatment in 8/35 patients (23%). Lesion detection by DHC was limited by tumour size and intensity of FDG uptake. Image fusion provided relevant clinical information in 9/35 patients (26%). CONCLUSION: Functional imaging in NSCLC with this dual-head gamma camera is superior to morphological imaging by CT, although inferior to dedicated PET imaging. Combined functional-anatomical imaging has the potential to improve staging and localization procedures before surgery or radiotherapy.

Adult↗
PubMed2004

Preoperative marking of musculoskeletal tumors guided by magnetic resonance imaging.

BACKGROUND: The purpose of this study was to evaluate a new stereotactic method for preoperative coil-marking of musculoskeletal tumors with use of interventional magnetic resonance imaging. METHODS: Nine patients with a soft-tissue or bone-marrow tumor were referred to our department for preoperative marking of the extent of the lesion. In one patient, two lesions were marked. Guidance for the punctures and the delivery of the coils was provided by an open low-field magnetic resonance imaging system with horizontal access. After imaging of the extent of the lesion, magnetic resonance imaging-compatible titanium coils were placed with use of nearly real-time or step-by-step magnetic resonance imaging control. The coils were placed up to seventy-two hours before the surgery. The inclusion of the tumor borders within the area of the excision was examined with cross-sectional histological analysis of surgical specimens. RESULTS: The tumor-marking intervention was successfully performed with the guidance of magnetic resonance imaging only in all patients. Preoperatively, nineteen coils were used to mark the ten lesions in the nine patients. All of the coils were easily located with intraoperative fluoroscopy. No coil migrated between the time of the percutaneous marking and the surgery. Histological examination of the resection borders revealed no residual tumor cells. No complications were observed, and, after a mean of twenty-three months of follow-up, no tumor had recurred. CONCLUSIONS: Preoperative coil-marking guided by magnetic resonance imaging for exact delineation of a musculoskeletal tumor is technically feasible and can readily demonstrate the full extent of the tumor. Use of magnetic resonance fluoroscopy reduces the time needed for the intervention. We recommend the coil-marking technique.

Adult↗
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↗