Wolfgang A. Wybranietz

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 cfrom 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.

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‐γ 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.—Deininger, M. H., Wybranietz, W. A., Graepler, F. T. C., Lauer, U. M., Meyermann, R., Schluesener, H. J. Endothelial endostatin release is induced by general cell stress and modulated by the nitric oxide/cGMP pathway. FASEB J. 17, 1267–1276 (2003)

A prototype transduction tag system (ΔLNGFR/NGF) for noninvasive clinical gene therapy monitoring

The dramatic expansion of clinical gene therapy trials requires the development of noninvasive clinical monitoring procedures, which provide information about expression levels, expression kinetics, and spatial distribution of transduced therapeutic genes. With the development of such procedures, invasive sampling of tissue probes from patients potentially could be reduced significantly. In this study, an experimental platform for the rational design and in vitro testing of suitable receptor-ligand couples as components of future transduction tag systems for noninvasive gene therapy monitoring applications was developed. Initially, the feasibility of the ΔLNGFR/nerve growth factor (NGF) transduction tag system was investigated; this system employs a mutated version of the low-affinity nerve growth factor receptor (p75mut or ΔLNGFR) lacking the entire cytoplasmic domain. Specific binding of 125I-radiolabeled NGF was demonstrated for two stable ΔLNGFR-transduced cell lines, but not for ΔLNGFR-negative parental control cell lines. An additional binding analysis performed in a MicroImager directly confirmed binding of radiolabeled ligands (125I-NGF, 125I-anti-p75 monoclonal antibody) to the p75mut expressed on intact target cells, but not on control cells. Subsequent binding studies employing NGF radiolabeled with the positron-emitting isotope 124I demonstrated a specific binding for LNGFR+ PC12 cells. Consequently, the first in vitro proof of a transduction tag approach based on the specificity of the 124I-NGF/LNGFR interaction was provided, which opens up the possibility for future noninvasive positron emission tomography monitoring in clinical gene therapy trials.

1013. Comparison of VP22 Intercellular Spreading in Live Versus Fixed Cells

Top of pageAbstract The property of the HSV-1 tegument protein VP22 of intercellular trafficking makes it a promising tool to overcome low transduction efficiencies in gene therapy. Over the last decade this distinctive ability of VP22 has been a prominent object of numerous investigations. However, recent reports not only proclaim (i) that VP22 cannot facilitate intercellular spreading, but (ii) that trafficking of VP22 fusion proteins results from artefacts of cell fixation only. To unravel this situation and to provide direct evidence for the presence or absence of VP22 mediated intercellular trafficking we generated an adenoviral vector with a dual expression cassette for VP22-GFP and DsRed both being under the control of distinct hCMV-promoters. Using this new vector type we were able to investigate not only the extent of VP22 mediated intercellular spreading by live fluorescence microscopy and FACS, but also onone and the same cells whether or not fixation (e.g., with paraformaldehyde) does influence VP22 mediated intercellular spreading. Our results clearly demonstrate that VP22-GFP can spread from less than 4% primary transduced cells to more than 70% cells of a cellular monolayer and that fixation with paraformaldehyde has no influence on VP22 mediated spreading. Thereby, we confirm the unique ability of VP22 mediated intercellular trafficking.

Mechanisms of viral induced caspase activation in human hepatoma cell lines

For a growing number of liver diseases like viral induced hepatitis or fulminant liver failure, apoptosis induction in hepatocytes plays a key role in the course of the disease. In the effector phase of apoptotic cell death a family of proteases, called caspases, is organized in a hierachial order, thus most members of this cascade are activated by other members being more upstream in the pathway. Therefore, investigation of the very first mechanisms leading to the activation of a so called initiator caspase like caspases-S or -9, are important steps in molecular diagnosis and treatment of various diseases. We have looked for new caspase activation pathways in various cell lines, including human hepatoma lines by infection in our model system with the paramyxovirus Sendai virus (SeV). We could show signs of apoptosis 15 to 20 h after SeV infection by TUNEL assay or FACS analysis of infected cells (e.g. HepG2). A direct influence of caspases on this process could be demonstrated by a complete inhibition of apoptosis by incubation of infected cells with the broad caspase inhibitor z-VADfmk. Subsequently we could not only detect the activation of caspase-3 which is regarded as an effector caspase being downstream of initiating events, but we could also demonstrate caspase-8 and -9 cleavage by immunoblotting in these cell lines. Previous work demonstrated caspase-S as the first caspase in the signal transduction via death receptors, whereas caspase-9 activation is regarded as the first activated caspase mediated by chemotherapeutic agents causing cytochrome c release from the mitochondria. Surprisingly neither the involvement of the death receptors CD95 or TNF-RI (inhibition experiments by decoy proteins), nor the release of cytochrome c (immunoblot, immunofluorescence) could be shown, indicating the existence of an alternative way of caspase-8 and/or caspase-9 activation. To further characterize this pathway we are currently investigating the involvement of other proteases, death receptors or even viral proteins in the activation of initiator caspases.