Institute for general pharmacology & toxicology -
Prof. Dr. J. Pfeilschifter

Expertise

Cancer:

Profound knowledge of virus-associated cancer types as models for developing tumor-specific therapies (EBV, HPV) (Virus-associated tumors in humans)
Identification of tumor suppressor regions and genetic lesions via monochromosome transfer, microsatellite polymorphism analysis and SSCP analysis
Improving strategies for cancer treatment: screening for synergistic effects in the combination of individual therapy regimens (prodrug converting enzyme systems as radiation sensitizers: use of specific antiviral therapeutics)
Design and evaluation of tumor-specific gene therapy vectors (based on adeno-, retro- or herpesviruses, maintained as artificial mini chromosomes; design of expression cassettes which depend on viral transcription factors or include promoters of aberrantly expressed genes; exploring the therapeutic potential of suicid genes, pro-apoptotic and tumor supressor genes)
Anti-cancer effects of analgetics (NSAIDs), development of tumor-specific delivery systems for these drugs to reduce side-effects
Tumorigenicity assays using immune-deficient mouse models; recultivation of tumor material in vitro

Inflammation:

In depth understanding of molecular mechanisms of kidney and skin inflammation
The Extracellular matrix as a target during inflammation: regulation of ECM degrading enzymes (matrix metalloproteinases (MMPs), (endogenous) plasminogen activators (PAs); investigating RNA stability as a posttranscriptional mechanism to regulate gene expression
Examination of inflammatory processes during tissue regeneration: cutaneous wound healing as a model system; effect of nitric oxide, type II diabetes, leptin, pharmacological intervention in the context of healing; keratinocyte biology
Cultures of human whole blood cells and of freshly isolated peripheral blood mononuclear cells as an established model to evaluate the production of pro- and anti-inflammatory cytokines in vitro
Human colon carcinoma cells as a model to investigate mechanisms of pro-inflammatory and pro-apoptotic activation
Investigating the role and localization of phospholipase A2 subtypes in healthy skin and in skin from patients with allergic or autommune diseases (phospholipase A2 activity assays (cytosolic and secretory) in vitro and in intact cells)

Signal Transduction:

Analysis of lipid mediators (radioactive, GC-MS, EIA)
Lipid analysis with TLC
Detection of PKC isoenzyme-mediated cellular functions (mesangial cells as model system; in vitro PKC isoenzyme activity assays)
Quantification of tetrahydrobiopterine
Quantification of the sphingolipids (also in human tissue) and identification of signaling pathways they are involved in
Identification of genes whose expression is impacted by nitric oxide: analysis of differential gene expression on RNA (RAP-PCR) and protein level

Resources and Abilities

Cloning, DNA sequencing, transformation / transfection, infection using viral vectors
FACS cell sorter
Virus production and concentration via ultracentrifugation
Immunohistochemistry
- Immunocytochemistry
- Immunofluorescence analysis of tissue sections (skin, kidney)
Confocal laser microscopy
Luciferase assays (promoter analysis, analysis of mRNA stabilizing elements via cloning of 3´ UTRs into reporter genes)
Electrophoretic mobility shift assays: DNA-/ RNA-protein interactions
Site-directed mutagenesis (functional analysis of distinct cis-regulating promoter elements)
In vitro RNA degradation assay (mRNA stability measurements)
Gelatin zymography (semiquantitative measurements of MMP-2 / MMP-9 activities from cell culture supernatants)
Differential gene expression analysis using 2D gel electrophoresis and differential display
In vitro models:
- Glomerular mesangial cells, MCF-7 breast cancer cells
- Cultures of human whole blood cells and of freshly isolated peripheral blood
- Human Burkitt lymphoma lines as model for virus-associated tumors
Primary mouse keratinocytes: isolation and cultivation from neonatalmicemononuclear cells
Human colon carcinoma cells: mechanisms of pro-inflammatory and pro-apoptotic activation (READ-OUT: modulation of cytokine expression, induction of apoptotic cell death)
Cultivation of primary human keratinocytes; cell lines derived from keratinocyte tumors
Animal models:
C57BL/6, BALB/c, db/db, ob/ob, transgene
nude and SCID mice

Publications

Westphal EM, Ge J, Catchpole JR, Ford M and Kenney SC: The nitroreductase / CB1954 combination in Epstein-Barr virus-positive B-cell lines: induction of bystander killing in vitro and in vivo. Cancer Gene Ther 7: 97-106, 2000.
Westphal EM, Blackstock W, Feng W, Israel B and Kenney SC: Activation of lytic Epstein-Barr virus infection by radiation and sodium butyrate in vitro and in vivo : a potential method for treating EBV-positive malignancies. Cancer Res 60: 5781-5788, 2000.
Feng WH, Westphal E, Mauser A, Raab-Traub N, Gulley ML, Busson P, Kenney SC: Use of adenovirus vectors expressing Epstein-Barr virus (EBV) immediate-early protein BZLF1 or BRLF1 to treat EBV-positive tumors. J Virol 76(21): 10951-9, 2002.
Akool E.S., Kleinert H., Hamada F.M.A., Abdelwahab M.H., Förstermann U., Pfeilschifter J, and Eberhardt W.: Nitric oxide increases the decay of MMP-9 mRNA by inhibiting the expression of mRNA stabilizing factor HuR. Mol. Cell. Biol. 23(14): 4901-4916, 2003.
Eberhardt W., Schulze M., Engels C., Klasmeier E. and Pfeilschifter J.: Glucocorticoid-mediated suppression of cytokine-induced matrix metalloproteinase-9 expression in rat mesangial cells: involvement of NF-kB and Ets transcription factors. Mol. Endocrinol. 16: 1752-1766, 2002.
Eberhardt W., Beck K.-F., Huwiler A., Walpen S., Pfeilschifter J.: Amplification of interleukin-1b-triggered MMP-9 expression in rat mesangial cells by superoxide is mediated by increased activity of NF-kB and AP-1 and involves activation of the MAPKs pathways. J. Immunol.165: 5788-5797, 2000.
Frank S, Stallmeyer B, Kämpfer H, Kolb, N, Pfeilschifter J: Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair. J Clin Invest 106:501-509, 2000
Goren I, Kämpfer H, Podda M, Pfeilschifter J, Frank S: Leptin and wound inflammation in diabetic ob/ob mice: differential regulation of neutrophil and macrophage influx and a potential role for the scab as a sink for inflammatory cells and mediators. Diabetes 52:2821-2832, 2003
Kämpfer H, Bräutigam L, Geisslinger G, Pfeilschifter J, Frank S: Cyclooxygenase-1-coupled prostaglandin biosynthesis constitutes an essential prerequisite for skin repair.J Invest Dermatol 120:880-890, 2003
Hellmuth M, Wetzler C, Nold M, Chang J-H, Frank S, Pfeilschifter J, Mühl H.: Expression of Interleukin-8, Heme Oxygenase-1, and Vascular Endothelial Growth Factor in DLD-1 Colon Carcinoma Cells Exposed to Pyrrolidine Dithiocarbamate. Carcinogenesis 23: 1273-1279, 2002.
Nold M, Hauser IA, Höfler S, Goede A, Eberhardt W, Ditting T, Geiger H, Pfeilschifter J, Mühl H.: IL-18BPa:Fc cooperates with immunosuppressive drugs in human whole blood. Biochem Pharmacol 66: 501-506, 2003.
Mühl H, Höfler S, Pfeilschifter J.: Inhibition of lipoplysaccharide/ATP-induced release of interleukin-18 by KN-62 and Glyburide. Eur J Pharmacol 482: 325-328, 2003.
Walpen S, Beck KF, Schaefer L, Raslik I, Eberhardt W, Schaefer RM, Pfeilschifter J:
Nitric oxide induces MIP-2 transcription in rat renal mesangial cells and in a rat model of glomerulonephritis. FASEB J. 15: 571-573, 2001
Schaefer L, Beck KF, Raslik I, Walpen S, Mihalik D, Micegova M, Macakova K, Schonherr E, Seidler DG, Varga G, Schaefer RM, Kresse H, and Pfeilschifter J.: Biglycan, a nitric oxide-regulated gene, affects adhesion, growth and survival of mesangial cells. J Biol Chem 278: 26227-26237, 2003

Staff:

PD Dr. W. Eberhardt (Extracellular matrix, inflammation)
PD Dr. S. Frank (Inflammation, wound healing, diabetes; in vivo test systems)
PD Dr. A. Huwiler (Signal transduction)
PD Dr. M. Kaszkin (Phospholipase A2, active substance analysis)
PD Dr. H. Mühl (Active substance analysis, signal transduction, inflammation)
Dr. K.-F. Beck (Nitric oxide-dependent gene expression, in vitro modl systems)
Dr. Eva-Maria Westphal (gene therapy; prodrug converting enzymes and NSAIDs as anti-cancer drugs; in vivo model systems)