Volume 43 (1997) No. 1
Volume 43 (1997) No. 1
In Memoriam
Flowers for George
D. Snell (1903-1996)......................1-3
Articles
Negative Regulation of the ð-Foetoprotein Gene in Fibroblasts: Identification and Characterization of cis and trans Elements
M.-F. HENRIETTE, P. GABANT, P.-L. DREZE, C. D. SZPIRER, J. SZPIRER..................................5-13
Département de Biologie Moléculaire, Université Libre de Bruxelles, Rhode-Saint-Genese, Belgium
Corresponding author: Claude Szpirer, Départment de Biologie Moléculaire, Université Libre de Bruxelles, Rue des Chevaux 67, B-1640 Rhode-Saint-Genese, Belgium. Tel. 32-2-650 9629, Fax 32-2-650 9625.
Abstract.
Flow Cytometric Analysis of Lymphocytes Isolated from Nasal Polyps
A. IHAN*, J. PODBOJ**, S. ŠUŠKOVIÈ***, A. CÖR+, M. MAJCEN*, E. MATOS*, N. GALE++, B. WRABER*.........................15-18
*Institute of Microbiology, Medical Faculty, Ljubljana, Slovenia
**Department of Otorhinolaryngology and Cervico-Facial Surgery, University Medical Centre, Ljubljana, Slovenia
***Institute of Pulmonary Diseases and Tuberculosis, University Medical Centre, Ljubljana, Slovenia
+Institute of Histology, Medical Faculty, Ljubljana, Slovenia
++Institute of Pathology, Medical Faculty, Ljubljana, Slovenia
Corresponding author: Alojz Ihan, Institute of Microbiology, Medical Faculty, University Ljubljana, Zaloska 7, 61 000 Ljubljana, Slovenia. Tel. (38661) 1403042; Fax (38661) 302 895; e-mail: IHAN@IBMI.MF.UNI-LJ.SI
Abstract.
Genotoxicity of N-Nitroso-N-Methylurea and Acetone Oxime in the Transgenic Drosophila Carrying the Human Gene Encoding a Subunit of Glutathione S-Transferase
M. RYŠKOVÁ*, K. CHROUST*, M. TRBUŠEK*, J. BENEDÍK*, T. JOWETT** .....................19-24.
*Department of Genetics and Molecular Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
**Department of Biochemistry and Genetics, Medical School, University of Newcastle, Newcastle upon Tyne, UK
Corresponding author: Karel Chroust, Department of Genetics and Molecular Biology, Faculty of Science, Masaryk Universite, Kotláøská 2, CZ-611 37, Czech Republic.
Abstract.
Treatment of Transplanted Spontaneous Rat T-Cell Leukaemia with Local Administration of Recombinant Murine Interleukin-2
B. OTOVÁ*, A. PANCZAK*, J. ŠÍMOVÁ**, T. JANDLOVÁ**, J. BUBENÍK**, K. BLAŽEK***, J. SCHRAMLOVÁ***, I. MARINOV+ ...................25-32
*Department of Biology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic
**Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
***National Institute of Public Health, Prague, Czech Republic
+Institute of Hematology and Blood Transfusion, Prague, Czech Republic
Corresponding author: Berta Otová, Department of Biology, 1st Faculty of Medicine, Charles University, Albertov 4, 128 00 Prague, Czech Republic.
Abstract.
Changes in Macrophage Function During Chemotherapy
D. VUÈKOVIC, M. DORIC...........................................................................................................33-38
Department of Microbiology, Medical Faculty, University of Rijeka, Rijeka, Croatia
Corresponding author: Darinka Vuèkovic, Department of Microbiology, Medical Faculty, University of Rijeka, Brace Branchetta 20/1, HR-51000 Rijeka, Croatia.
Abstract
Short Communications
Recombinant Interleukin-2 Acts as an Adjuvant and Helps to Increase the Efficacy of Tumour Vaccines
P. VONDRYS, J. ŠÍMOVÁ, Š. TAKÁÈOVÁ, T. JANDLOVÁ, J. BUBENÍK ...............39-40
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 166 37 Prague, Czech Republic
Corresponding author: Dr. Jan Bubeník, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 37 Prague 6, Czech Republic.
Abstract
Putative Regulatory Sequence in Human Papillomavirus Type 16 E2 Open Reading Frame
H. KITASATO*, A. TARDY-KADLEC*, M. ŠMAHEL*, R. TYKVA**, V. VONKA* ..............41-44
*Department of Virology, Institute of Hematology and Blood Transfusion, Prague, Czech Republic
**Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Corresponding author: Michal Šmahel, Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20 Prague 2, Czech Republic. Fax +42-2-291073; e-mail: smahel@hema.uhkt.anet.cz.
Abstract
CTLL Assay: Comparison of Two Methods for IL-2 Determination M.
INDROVÁ*, E. PAJTASZ-PIASECKA**, C. RADZIKOWSKI**, J. BUBENÍK* ..........................5-47
*Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic **Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
Corresponding author: Dr. Marie Indrová, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 37 Prague 6, Czech Republic.
Abstract
Epitope Analysis of the Human p53 Tumour Suppressor Protein
H. DOLEŽALOVÁ, B. VOJTÌŠEK, J. KOVAØÍK......................49-51
Department of Cellular and Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
Corresponding author: Hana Doležalová, Department of Cellular and Molecular Oncology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic
Abstract
Abstracts
Negative Regulation of the ð-Foetoprotein Gene in Fibroblasts: Identification and Characterization of cis and trans Elements
M.-F. HENRIETTE, P. GABANT, P.-L. DREZE, C. D. SZPIRER, J. SZPIRER
The ð-foetoprotein (AFP) gene is extinguished in hybrids formed between hepatoma cells (expressing cells) and fibroblass (non-expressing cells). Tranfection experiments with constructions containing segments from the promoter region of the AFP gene, placed upstream of an ubiquitously expressed promoter (the Herpes virus thymidine kinase gene promoter), showed that the AFP gene-derived sequence contains at least one negative element active in fibroblasts (while this sequence behaves as an enhancer in hepatoma cells). We identified such a fibroblast negative region, localized between nucleotide positions -80 to -38 (FNE1). Gel retardation experiments showed that FNE1 specifically binds fibroblast nuclear proteins, generating three complexes. The sequence from -57 to -43 was shown to be responsible for both the formation of these complexes and the negative activity of FNE1. These results suggest that the binding of nuclear factors to the AFP promoter region contributes to silencing the AFP gene in non-expressing cells, such as fibroblasts, and thus to establishing lineage-specific expression of the AFP gene.
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Flow Cytometric Analysis of Lymphocytes Isolated from Nasal Polyps
A. IHAN*, J. PODBOJ**, S. ŠUŠKOVIÈ***, A. CÖR+, M. MAJCEN*, E. MATOS*, N. GALE++, B. WRABER*
We analyzed flow cytometrically the expression of IL-2 receptors (ð subunit - CD25) and ICAM-1 adhesion molecules (CD54) on T cells and subsets (CD4, CD8) isolated from nasal polyp tissue in allergic and non-allergic patients. We found a significant increase in IL-2 receptor and ICAM-1 molecule expression on T cells isolated from nasal polyp tissue compared to peripheral blood lymphocytes. We also found a significantly increased expression of ICAM-1 molecules on CD8+ cells in non-allergic compared to allergic patients. The latter may reflect a difference in cytotoxic immune response between allergic and non-allergic patients, but the result should be confirmed in a more extensive study including cytokine and immunoglobulin analysis. We hope that it would enable us to obtain a deeper insight into the local immune events and further to clarify the aetiology and pathogenesis of nasal polyps and their relation to allergy.
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Genotoxicity of N-Nitroso-N-Methylurea and Acetone Oxime in the Transgenic Drosophila Carrying the Human Gene Encoding a Subunit of Glutathione S-Transferase
M. RYŠKOVÁ*, K. CHROUST*, M. TRBUŠEK*, J. BENEDÍK*, T. JOWETT**
The genotoxic effects of N-nitroso-N-methylurea (MNU) and acetone oxime (ACOX) were tested in the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. We have performed the same assay on transgenic flies expressing the human gene encoding a glutathione S-transferase alpha subunit (HGST). The SMART assay is used here to demonstrate genotoxicity and to determine the effect of human glutathione S-transferase on the genotoxic response. Three types of Drosophila strains were used: non-transgenic strain first described by Szabad (1986), transgenic strains derived from the Szabad strains but expressing the bacterial lacZ gene, and similarly derived transgenic strains expressing the HGST gene. MNU was highly genotoxic in both transgenic and non-transgenic flies. The non-transgenic flies were significantly more sensitive to the genotoxic effects of MNU compared to both types of transgenic flies. There were statistically significant differences between the transgenic HGST crosses and transgenic lacZ and non-transgenic control crosses but there was no significant difference between the genotoxic response to MNU in flies from the transgenic cross with lacZ and from the cross carrying three copies of HGST. ACOX also proved to be genotoxic to both non-transgenic and transgenic flies. However, flies carrying three copies of the gene were significantly more resistant to the genotoxic effect of ACOX than those transgenic flies with two or no copies of the human gene.
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Treatment of Transplanted Spontaneous Rat T-Cell Leukaemia with Local Administration of Recombinant Murine Interleukin-2
B. OTOVÁ*, A. PANCZAK*, J. ŠÍMOVÁ**, T. JANDLOVÁ**, J. BUBENÍK**, K. BLAŽEK***, J. SCHRAMLOVÁ***, I. MARINOV+
Spontaneous rat CD4+CD8- T-cell leukaemia transplanted in syngeneic recipients served as an experimental model system for IL-2 therapy. As a source of IL-2, supernatants from in vitro cultured plasmacytoma cell line X63-m-IL2 secreting constitutively recombinant murine IL-2 were utilized. Administration of IL-2, s.c. to the vicinity of the tumour inoculum, suppressed tumour growth. The tumour-inhibitory IL-2 effects were time- and dose-dependent. When the treatment has started 10 days after the challenge with 104 leukaemia cells, IL-2 inhibitory effects on the lymphoma growth in situ were demonstrated by lower tumour weight combined with necrotic changes. No histological signs of lymphoma generalization were found in parenchymatous organs of IL-2-treated rats in contrast to the untreated controls. No histological or functional injuries to the kidneys due to IL-2 administration were found. The results of effector cell phenotyping demonstrated the kinetics of the CD4+/CD8+ ratio characterized by CD4+ T-cell depletion and resulting increase in the percentage of CD8+ PBL.
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Changes in Macrophage Function During Chemotherapy
D. VUÈKOVIC, M. DORIC
Antibiotics, in addition to killing or inhibiting the growth of microorganisms, may also affect the mechanism of host defence in many ways. Such effects may be clinically relevant especially in the case where an impairment of immunological function can be seen. We, therefore, decided to study the influence of penicillin G, cefotaxim, ceftazidime, streptomycin, and lincomycin on the function of phagocytes by using the macrophage adherence assay and the macrophage spreading assay. We also followed the concentrations of neopterin and interferon gamma (IFN-gamma) in the plasma of mice treated with the above mentioned antibiotics. Changes in adherence of peritoneal macrophages were seen in mice treated with therapeutic doses of penicillin G and cefotaxim, after 2 h of incubation. Cefotaxim and streptomycin in the usual therapeutic dose and ceftazidime in a fourfold higher dose influenced the capacity of peritoneal macrophages to spread on a glass surface. The same was seen with lincomycin when administered in the therapeutic dose and in a fourfold higher dose. In all the mice treated with antibiotics the concentration of IFN-gamma was higher than in the control mice, while the reverse was seen concerning neopterin release, with an exception in mice treated with streptomycin.
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Recombinant Interleukin-2 Acts as an Adjuvant and Helps to Increase the Efficacy of Tumour Vaccines
P. VONDRYS, J. ŠÍMOVÁ, Š. TAKÁÈOVÁ, T. JANDLOVÁ, J. BUBENÍK
To examine augmentation of the resistance-inducing effect of tumour vaccines with IL-2, we have used the cytokine produced by genetically modified somatic cells, and a conventional experimental model of TRA-expressing, MC-induced murine fibrosarcoma transplanted in histocompatible mice. We have found that the effect of s.c. immunization with irradiated tumour vaccines can be substantially enhanced by IL-2 injected repeatedly at the site of vaccination. To investigate the kinetics of local and systemic immunocyte populations during the course of immunization with vaccine plus IL-2, some of the vaccinated mice were sacrificed and their regional LNC and spleen cells were used for phenotypic analysis. It has been found that local (s.c.) administration of the irradiated tumour vaccine plus IL-2 was accompanied by an early depletion of CD4+ and CD8+ cells in regional lymph nodes followed by subsequent rebound lymphocytosis. The local reaction was followed by a systemic response in the spleen characterized with an increase in TCRðÐ+ CD4+ lymphocytes.
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Putative Regulatory Sequence in Human Papillomavirus Type 16 E2 Open Reading Frame
H. KITASATO*, A. TARDY-KADLEC*, M. ŠMAHEL*, R. TYKVA**, V. VONKA*
A 114bp fragment of the human papillomavirus type 16 (HPV16) E2 open reading frame (nt. 3142-3255) containing a putative estrogen responsive element (ERE) was amplified and cloned into pBLCAT2 plasmid in both sense (p159-4) and anti-sense (p164) orientation. The plasmids were transfected into human breast-cancer cell line MCF-7 containing estrogen receptor and the cultures were kept in the presence or absence of b-estradiol. The chloramphenicol acetyltransferase (CAT) activity was not influenced by estrogen. However, a silencer effect was observed both in cultures transfected with p159-4 and p164 plasmids. We prepared and cloned synthetic fragments containing the putative ERE and failed to prove that the palindrome in the putative ERE was responsible for the silencer activity.
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CTLL Assay: Comparison of Two Methods for IL-2 Determination M.
INDROVÁ*, E. PAJTASZ-PIASECKA**, C. RADZIKOWSKI**, J. BUBENÍK*
Two different modifications of CTLL techniques for IL-2 detection, the routine 3H-thymidine incorporation assay and the colorimetric MTT method, were compared. The reproducible results of the studies of supernatants from in vitro propagated transfected cells were obtained. The employed methods appeared to be comparable and are used simultaneously in our cooperating laboratories for estimation of the levels of IL-2 secreted by the transfectants that are applied as the source of the cytokine in therapeutic experiments.
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Epitope Analysis of the Human p53 Tumour Suppressor Protein
H. DOLEŽALOVÁ, B. VOJTÌŠEK, J. KOVAØÍK
Using a set of overlapping peptides of the human p53 protein, we have performed an accurate mapping of the p53 antigenic sites, recognized by a panel of 19 monoclonal antibodies from the Bp53 series. The results show that most of the antibodies recognize determinants localized in the amino-terminal domain of the protein. Several antibodies reacted with peptides which correspond to the antigenic determinants localized in the carboxy terminus of p53. None of these antibodies reacted with peptides in the central DNA-binding domain of p53 protein.
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