13th Heidelberg Cytometry Symposium

Oral presentations (alphabetical order) Interferon-gbeeinflußt den Apoptose-Signalweg von TNFa nur teilweise über NFkB in der humanen Tumor Zell-Linie Me-180 Heinz BAISCH Institut für Biophysik und Strahlenbiology, Universitäts Klinikum Hamburg-Eppendorf In Me-180 Zellen induziert TNFa Apoptosis. Die Zellen lösen sich vom Kulturflaschenboden und zeigen apoptotische Charakteristika wie Chromatin-Kondensantion, Mitochondrien Membran Depolarisation, Phosphatidyl-Serin Exposition, PARP-Spaltung. Interferon g (IFNg) induziert fast keine Apoptosis, die Kombination von TNFa und IFNg ergibt jedoch einen dramatischen Anstieg des Anteils apoptotischer Zellen. Das deutet auf ”crosstalk” zwischen den TNFaand IFNg-Signalwegen hin. Durch TNF werden pround anti-apoptotische Signale ausgelöst. Ein anti-apoptototischer Weg führt über den Transkriptionsfaktor NFkB. Vom IFNg ist bekannt, dass es über STAT-1 die Expression von Inhibitoren (z.B. p202) der Transkriptionsfaktoren AP-1 and NFkB aktiviert. Dies könnte als Mechanismus, der die TNF-induzierte Apoptose durch IFN verstärkt, in Frage kommen. Wir haben die NFkB Veränderung im Zellkern nach TNF und IFN untersucht. Mit Antikörpern gegen NFkB haben wir eine kurzfristige Erhöhung nach TNF und Absenkung nach IFN nicht gefunden. Allerdings deutete ein Abbau von ikB nach TNF auf eine Aktivierung von NFkB hin. Dies wurde mit transienter Transfektion von Plasmiden, die für ikB-Grün-Fluoreszenz-Protein Fusionsprotein kodierten, gemessen. Nach 1 bis 3 Tagen TNFEinwirkung war NFkB im Zellkern etwa doppelt so hoch wie in der Kontrolle, während IFN keine Veränderung gegenüber der Kontrolle bewirkte. Durch die gemeinsame Gabe von IFN und TNF konnte der NFkB-level nicht gegenüber TNF allein abgesenkt werden. Diesen nicht ganz dem Modell entsprechenden Ergebnisse stehen Resultate aus Messungen mit NFkB-Inhibitoren gegenüber. Mit einer Reihe verschiedener Inhibitoren konnte durchgehend der Apoptose-Anteil von TNF allein erhöht werden, wenn sie in Kombination mit TNF gegeben wurden. Allerdings wurde in keinem Fall eine so starke Steigerung der Apoptose wie durch IFN erreicht. Insgesamt zeigen unsere Ergebnisse, dass NFkB in dieser Zelllinie nur zu einem geringen Teil der Wechselwirkung zwischen den IFNgund TNFa-Signalwegen zu Grunde liegt. Detection of Membrane Glucocorticoid-Receptor Expression on Peripheral Blood Monocytes Using High-Sensitivity Immunofluorescence Burkhard Bartholome$, Désirée Kunkel*, Maren Bienert$, Timea Berki§, Alexander Scheffold*, and Frank Buttgereit$ $ Charité, University Clinics of the Humboldt-University Berlin, Schumannstr. 21/22, 10117 Berlin; §Department of Immunology and Biotechnology, University Medical School of Pécs, Hungary; *Deutsches Rheuma-Forschungszentrum Berlin, Schumannstr. 21/22, 10117 Berlin Glucocorticoid receptors (GCR) can easily be detected by conventional immunofluorescence in the cytoplasm but not on the surface of many different cell types. Recent data provide evidence that GCR are expressed at low level on the surface of certain leukemia cell lines. We have used high-sensitivity immunofluorescent staining (magnetofluorescent liposomes) to analyze the expression of GCR on cell lines and normal peripheral blood mononuclear cells (PBMC). We compared the frequency of GCR positive cells in PBMC from vaccinated persons, Rheumatoid Arthritis patients, and healthy controls. Basic GCR expression (5-6%) could be demonstrated on monocytes from healthy controls. However, healthy people following vaccination and patients with active Rheumatoid Arthritis have clearly elevated level of GCR expression on peripheral blood monocytes (up to 20%). The expression level also correlates with clinical parameters of disease activity. 13th Heidelberg Cytometry Symposium file:///M:/cytometry/abstracts-13HCS.htm 2 von 27 16.3.2005 09:53 This data clearly demonstrate GCR surface expression on normal human peripheral blood monocytes. Furthermore the expression level seems to correlate with an activated immune system, e.g. in chronic autoimmune disease or following vaccination. Cell Cycle and Growth Response of CHO Cells to Low Doses of X-Rays D. Bartkowiak, S. Hogner, W. Nothdurft, E.M. Rottinger Universitat Ulm Klinikum, Abteilung Strahlentherapie, Robert-KochStr.6, D-89081 Ulm, Germany This study was stimulated by reports of hyperradiosensitivity (HRS ) of various cell lines to very low X-ray doses. HRS could have implications in both, clinical and environmental exposure. It has be en interpreted as a result of a threshold for the induced repair ( IR) of DNA damage. Survival data of exponentially growing X-irradiated Chinese hamster ovary cells were acquired by conventional colony forming assay and by flow cytometric population counting. The data were subjected to best-fit analyses with both, the linear-quadratic (LQ) and the IR model. Flow cytometric DNA and DNA/BrdU measurements served to test cell cycle reactions for compatibility with HRS. Both, the LQ and the IR model sufficiently describe survival curves established by colony forming assays. In contrast, population counting data were exactly and exclusively in accordance with the LQ model. Four hours after irradiation, cell cycle distributions showed a dose dependent mitotic block, delay of cells in G2 and, after higher doses, increasingly in late S-phase. All alterations were monotonous between 0.2 and 3 Gy. No convincing evidence of HRS was found in these experiments. For survival data, the LQ model applies to the full dose range from 0.2 to 7 Gy. The sensible cell cycle response is incompatible with an "ignorance" of the cells even to low amounts of DNA damage. More likely, they reflect an alert condition, supporting the assumption of a threshold-free IR. Multiparameter Analysis of Progeny of Individual Cells by Laser Scanning Cytometry (LSC) Elzbieta Bedner1,2, and Zbigniew Darzynkiewicz1 1Brander Cancer Research Institute, New York Medical College, Hawthorne, N.Y. 10532, USA 2Department of Pathology, Pomeranian School of Medicine, Szczecin, Poland LSC was adapted to analyze size and phenotype of colonies of MCF-7 cells growing in microscope slide chambers, untreated and treated with the cytotoxic ribonuclease, onconase (ONC). Data representing each colony was segmented based on BODIPY 630/650-X fluorescence (> 650 nm) excited by a He-Ne laser. The cells’ DNA stained with PI and estrogen receptor (ER) or p53 (FITC) were detected by Ar ion laser excitation. The following attributes of individual colonies were measured: (a) area, circumference, area/circumference ratio, (b) DNA or protein content/colony area, (c) number of cells (nuclei), (d) DNA content and cell cycle distribution, (e) protein content and protein/DNA ratio, and (e) expression of ER or p53 per colony, per total protein, per nucleus or per DNA, within a colony. Heterogeneity of colonies with respect to all the measured features was estimated. Changes in colony size and phenotype reflecting altered cell shape, size, colony protein/DNA ratio, expression of individual proteins, may reveal mechanisms of drug action to suppress proliferative capacity of the cells, and detect growth imbalance, differentiation and modulating expression of the genes that may be associated with cell cycle, apoptosis or differentiation. With minor modifications LSC may be applicable for automatic analysis of cloning efficiency and multiparameter analysis of cell colonies in soft agar. Such analyses may be useful in studies of mechanisms and effectiveness of antitumor drugs, in field of carcinogenesis, for analysis of primary cultures, tumor prognosis and drug sensitivity and in analysis of microbial colonies. Apoptosis and Caspase Activity in Peripheral Blood Lymphocytes of Patients with Lupus Erythematosus Detected by Flow Cytometry I. Böhm Department of Dermatology, University of Bonn, Germany Cells undergoing apoptosis can be detected flow cytometrically be a variety of different methods including annexin V assay, disruption of mitochondrial membrane potential (??m), TUNEL Assay, detection of 13th Heidelberg Cytometry Symposium file:///M:/cytometry/abstracts-13HCS.htm 3 von 27 16.3.2005 09:53 poly(ADP)-ribose-polymerase fragmentation, and sub-G1-peak for example. Sequential activation of initiator and effector-caspases (cysteine proteases with aspartic acid specificity) is a biochemical hallmark of cells undergoing apoptosis. Commonly used techniques for the detection of activated caspases in different cell systems are western blot analysis, colorimetric and fluorometric assays. These are time consuming methods and additionally have the disadvantage that a simultaneous labelling with specific mAbs is not possible. To overcome these problems in the present preliminary investigation we used covalently to rhodamine 110 [(L-Asp)2-rhodamine 110] bound aspartyl compounds as pan-caspase substrate. The complete complex is colorless, but after caspase-induced cleavage the fluorochrome is released and can be detected either by flow cytometry, fluorescence microscopy or laser scanning microscopy (excitation 488 nm, emission 515 545 nm). Since patients with lupus erythematosus display an increased in vivo frequency of blood cells undergoing apoptosis, we analyzed peripheral lymphocytes of these patients. Freshly obtained whole blood samples were compared to density gradient centrifugation isolated PBMC. Additionally, apoptosis was measured by using the annexin V assay. We could find caspase activity in peripheral lymphocytes of patients with LE as compared to healthy controls with this technique. (L-Asp)2-rhodamine staining was possible in PBMC as well as in whole blood samples. Annexin V Labelling of membrane phosphatidylserine exposure did not correlate with (L-Asp)2-rhodamine cleavage. Taken together, the obtained results show that (L-Asp)2-rhodamine 110 is an interesting tool for the flow cytometrically based investigation of pan-caspase activity, because (i) it is a rapid method suitable to screen pan-caspase activity on single cell level, and (ii) it can be used with simultaneous mAb staining. Further investigations to confirm this observation and clarify it in more detail are currently underway. Objective Grading and Prognostification of Dysplasias of the Uterine Cervix Using a Combination of Laser Scanning Cytometry and HPV-PCR. An one Step Method. Bollmann, R.; Schmitz, J.M.; Speich N.; Vogel, C.U.; Schmitt C.; Bollmann, M. Institut f. Pathologie Heilsbachstr.15 D-53123 Bonn-Duisdorf, Germany Grading of cervical dysplasias as a morphological method is subjective. DNA aneuploidy is regarded as the starting point of cervical carcinogenesis. Aneuploidy can be measured by interactive image cytometry after Feulgen staining. Image DNA-cytometry of pap-smears after Feulgen staining has proved as an objective method of classification: DNA diploid dyplasias are low risk lesions (LSIL), DNA aneuploid dysplasias are high risk lesions (HSIL). Image cytometry though only can be done as a second step next to screening the stained pap-smears to render a diagnosis. 1The newly developed Laser scanning cytometer (LSC) in combination with 1fluid cytology makes it possible to perform simultaneously cytological diagnosis, DNA ploidy measurement and HPV-typing . Material and methods: From 22 patients with known SIL ("III-D") we performed monolayer preparations using the ThinPrep-Paptest (Cytyc). DNA ploidy was measured after PI staining with the LSC (Compucyte). Subsequently the smears were stained according to Papanicolaou and visualisized and classified after automatic relocalisation regarding to the DNA content und coordinates. Simultanously we performed detection and typing of HPV by PCR and direct sequencing.