Volume 45 (1999) No. 5

Editorial
Genomics on the Eve of the 21^st Century
V. PACES...........................167
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Corresponding author: Václav Pačes, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ16637 Prague, Czech Republic. Tel. (420-20) 20183541. Fax (420-2) 24311019. E-mail: vpaces@img.cas.cz.

Articles
Granulocyte-Macrophage Colony-Stimulating Factor-Producing Tumour Vaccines
P. RÖSSNER, J. BUBENÍK, V. SOBOTA, M. INDROVÁ, R. HÁJKOVÁ, L. MENDOZA,
T. JANDLOVÁ, J. ŠÍMOVÁ........................173
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague,
Czech Republic
Corresponding author: Jan Bubeník, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 37 Prague 6, Czech Republic.
Abstract.

Monitoring of Residual Disease in Non-Hodgkin's Lymphomas by Quantitative PCR (Preliminary Report)
A. SLAVÍČKOVÁ, V. ULLMANNOVÁ, E. BENEŠOVÁ, P. KLENER...................179
1^st Department of Internal Medicine, 1^st Faculty of Medicine, Charles University,
Prague, Czech Republic
Corresponding author: Alena H. Slavíčková, Laboratory of Molecular Biology, 1st Department of Internal Medicine, Faculty General Hospital, U Nemocnice 2, 128 08 Prague 2, Czech Republic.
Abstract.

Selective Cytotoxic Activity of a Novel Ribonucleoside Diphosphate Reductase Inhibitor MDL-101,731 against Thyroid Cancer in vitro
R. KOTCHETKOV^1,3, A. A. KRIVTCHIK³, J. CINATL¹, B. KORNHUBER¹,
J.CINATL², Jr.........................................................................................185
¹ Pediatric Clinic for Hematology and Oncology, and ² Institute for Medical Virology, University Clinics of Johann Wolfgang Goethe University, Frankfurt am Main, Germany,
³ Department of Pathophysiology, Medical Academy, Minsk, Belarus
Corresponding author: Ruslan Kotchetkov, Institut für Medizinische Virologie, Zentrum der Hygiene, Klinikum der Johann Wolfgang Goethe-Universität, Sandhof-Str. 2-4, 60528, Frankfurt am Main, Germany. Tel.: +49-69-6301-3943; Fax: +49-69-6301-4302; E-mail: kotchetk@stud.uni-frankfurt.de.
Abstract.

Aggregated forms of heparin-binding and non-heparin-binding proteins of boar seminal plasma and their binding properties
P. MAŇÁSKOVÁ^1,2, A. MÉSZÁROSOVÁ¹, J. LIBERDA², Z. VOBURKA³, M. TICHÁ²,
V. JONÁKOVÁ¹.......................................................................................193
¹Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
²Department of Biochemistry, Charles University, Prague, Czech Republic
³Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Prague, Czech Republic
Corresponding author: Věra Jonáková, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 37 Prague 6, Czech Republic. Fax: 420 2 24310955, Tel: 420 2 20183347, E-mail: vjon@img.cas.cz.
Abstract.

HXB/Ipcv and BXH/Cub Recombinant Inbred Strains of the Rat: Strain Distribution Patterns of 632 Alleles
M. PRAVENEC^1,2, V. KŘEN^1,2, D. KŘENOVÁ², V. BÍLÁ², V. ZÍDEK¹, M. ŠIMÁKOVÁ¹,
A. MUSILOVÁ¹, H. A. VAN LITH³, L. F. M. VAN ZUTPHEN³.....................................203
¹Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
²Institute of Biology, 1st Medical Faculty, Charles University, Prague, Czech Republic
³Department of Laboratory Animal Science, Veterinary Faculty, Utrecht University, Utrecht,
The Netherlands
Corresponding author: Michal Pravenec, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic. Phone/Fax: (4202) 475 2297; e-mail: pravenec@biomed.cas.cz.
Abstract.

Local Hypertrophic/Hyperplastic Changes of Keratinizing Squamous Epithelium of Pinna Induced by Concanavalin A and Other Immunomodulators in Mice
K. WLODARSKI¹, M. LUCZAK², R. GALUS¹, P. WLODARSKI¹...............................217
¹Department of Histology and Embryology, Institute of Biostructure, Medical Academy, Warsaw, Poland
²Department of Medical Microbiology, Institute of Biostructure, Medical Academy, Warsaw, Poland
Corresponding author: Krysztof Wlodarski, Department of Histology and Embryology, Institute of Biostructure, Medical Academy, 02-004 Warszawa, Chalubinskiego 5, Poland.
Abstract.

Monoclonal Antibody Register
A Set of Monoclonal Antibodies Specific for Bovine Cell Surface Molecule CD9
J. TOMÁŠKOVÁ, R. DUŠINSKÝ, L'. HOROVSKÁ, M. SIMON..................................225
Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovakia
Corresponding author: Jana Tomášková, Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, 900 28 Ivanka pri Dunaji, Slovakia. Tel: +421 (0)7 45943 882; fax: +421 (0)7 45943 932; e-mail: tomaskova@ubgz.savba.sk.
Background.

Articles
Granulocyte-Macrophage Colony-Stimulating Factor-Producing Tumour Vaccines

P. RÖSSNER, J. BUBENÍK, V. SOBOTA, M. INDROVÁ, R. HÁJKOVÁ, L. MENDOZA,
T. JANDLOVÁ, J. ŠÍMOVÁ

Murine sarcoma MC12 cells were transfected with the gene coding for murine granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumorigenicity of a variety of cell clones with different expression of the inserted gene was assessed. All of the genetically manipulated MC12 cell clones examined were found to be less tumorigenic than the parental MC12 cell population. No correlation was observed between the production of GM-CSF by the clones and their tumorigenicity. It has been found that irradiation of the GM-CSF-producing cells with the dose of 150 Gy did not significantly inhibit the GM-CSF production during the period of 5 days after irradiation. These findings provided us with the rationale for using the irradiated GM-CSF-producing MC12 sarcoma vaccine for therapy. It has further been found than immunosensitivity of the genetically manipulated, GM-CSF-producing tumour targets to the IL-2-activated killer (LAK) cell-mediated cytolysis was significantly increased, as compared to the parental target cell population. Irradiated, GM-CSF-producing tumour vaccines were used for therapy of 3-day-old MC12 sarcoma transplants in syngeneic mice and for therapy of surgically induced minimal residual tumour disease. Neither small tumour transplants, nor tumour residua after surgery were significantly sensitive to the therapy with GM-CSF-producing tumour vaccines.
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Monitoring of Residual Disease in Non-Hodgkin's Lymphomas by Quantitative PCR (Preliminary Report)
A. SLAVÍČKOVÁ, V. ULLMANNOVÁ, E. BENEŠOVÁ, P. KLENER

Highly sensitive PCR techniques are often used in molecular monitoring of hematological malignancies, and a quantification of residual disease is important for further prognosis. Here, the limiting dilution methodology and the multiplex IgH/ras PCR are proposed as approaches to molecular monitoring of NHLs.
Applying the limiting dilution methodology as a simple dose-response assay for the translocation t(14,18) and CDR3 clonal rearrangement of IgH, critical amounts of total cells determined with stored consecutive diagnostic samples in the same PCR run are compared. Assuming that specific targets are diluted proportionally in dilution of total genomic DNA, the samples showing lower critical concentrations of total DNA are considered as containing higher portion of cells possessing the specific disease marker and vice versa. So far, the correlation of results with the disease outcome confirmed that this simple semi-quantitative approach may in some cases substitute laborious precisely quantifying techniques in the monitoring of the disease.
In optimized multiplex IgH/ras PCR co-amplifying clonal CDR3 rearrangement of IgH and the codon 61 of Hras 1 gene, the amount of CDR3 product as the disease marker is related to the ras product as a standard marker of all cells, and quantitative results are obtained by software analyses of detecting gels.
Presumably, both approaches may provide clinically useful information on the disease activity and treatment outcome.
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Selective Cytotoxic Activity of a Novel Ribonucleoside Diphosphate Reductase Inhibitor MDL-101,731 against Thyroid Cancer in vitro

R. KOTCHETKOV, A. A. KRIVTCHIK, J. CINATL, B. KORNHUBER, J.CINATL, Jr.

The dramatically increased number of new cases of disseminated children thyroid cancer in Belarus after the Chernobyl accident requires a development of novel strategies to treat this disease. In addition to conventional therapy using radiojodine, the chemotherapy with new effective drugs can be an alternative kind of treatment. We tested the antitumor activity of (E)-2'-fluoromethylene-2'-deoxyci tidine (MDL-101,731), a ribonucleoside diphosphate reductase inhibitor against three thyroid carcinoma cell lines established from anaplastic (8505C), papillary (B-CPAP) and poorly differentiated papillary (BHT-101) cancer. MDL-101,371 decreased both cell growth and DNA synthesis of tumor cells at concentrations lower than 100 nM while the concentrations higher than 5000 nM showed only moderat effects on growth of normal human fibroblasts. The effects of MDL-101,371 on tumor cells were associated with induction of apoptosis as demonstrated by DNA fragmentation assay. The flow cytometric analysis of expression of apoptosis-related genes revealed increased levels of Fas-antigen whereas levels of Bcl-2 were not significantly influenced in thyroid cancer cells treated with MDL-101,731. These results demonstrated that MDL-101,731 is a potent antitumor agent against cultured thyroid cancer cells due to its ability to induce apoptosis in association with increased Fas expression.
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Aggregated forms of heparin-binding and non-heparin-binding proteins of boar seminal plasma and their binding properties

P. MAŇÁSKOVÁ, A. MÉSZÁROSOVÁ, J. LIBERDA, Z. VOBURKA, M. TICHÁ, V. JONÁKOVÁ

Boar seminal plasma proteins were separated by affinity chromatography on immobilized heparin into two portions: heparin-binding (H+) and non-heparin-binding (H) proteins. Gel chromatography of the H+ portion yielded four main protein fractions of >100, 45, 30 and 20 kDa while that of the H portion resulted in the separation into three main protein fractions of >100, 30 and 20 kDa. HPLC analysis and N-terminal sequencing used to characterize the composition of the protein fractions obtained by gel chromatography revealed that all consisted of low (12--16 kDa) molecular weight components: the H+ fraction consisted of DQH sperm protein, AQN and AWN spermadhesins whereas the H fraction consisted of PSPI and PSPII spermadhesins. The high molecular weight values of fractions obtained by gel chromatography thus suggest that the proteins are present in boar seminal plasma in the form of aggregates. Interactions of individual boar seminal plasma proteins and their aggregates present in the H+ and H fractions with acid polysaccharides were estimated.
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HXB/Ipcv and BXH/Cub Recombinant Inbred Strains of the Rat: Strain Distribution Patterns of 632 Alleles

M. PRAVENEC, V. KŘEN, D. KŘENOVÁ, V. BÍLÁ, V. ZÍDEK, M. ŠIMÁKOVÁ, A. MUSILOVÁ, H. A. VAN LITH, L. F. M. VAN ZUTPHEN

The HXB/Ipcv and BXH/Cub sets of recombinant inbred (RI) strains were derived from the spontaneously hypertensive rats (SHR/OlaIpcv) and normotensive Brown Norway (BN-Lx/Cub) rats. The RI strains were produced as a model system for genetic and correlation analysis of spontaneous hypertension and other risk factors of cardiovascular disease such as insulin resistance and dyslipidemia. The RI strains were phenotyped in multiple hemodynamic and metabolic traits. In the current study, we describe strain distribution patterns of 631 genetic markers.
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Local Hypertrophic/Hyperplastic Changes of Keratinizing Squamous Epithelium of Pinna Induced by Concanavalin A and Other Immunomodulators in Mice

K. WLODARSKI, M. LUCZAK, R. GALUS, P. WLODARSKI

Intradermal administration of concanavalin A, a potent T-cell mitogen, into an ear lap resulted in activation of chondrogenesis and stimulation of epidermis proliferation. This proliferation is sometimes invasive in character (pearls and epidermal nests form in the underlying connective tissue) but never turns into true cancerous lesions. This reaction can be delayed, but not prevented, by the prostaglandin inhibitor indomethacin.
Stimulation of epidermis proliferation was also caused by administration of other immunomodulators, such as carrageenan type IV, Moloney sarcoma development, and rarely in the course of GvHr, but to much lesser degree than with concanavalin A.
It is suggested that the same growth factors, which are mediators of local chondrocyte stimulation, are also mediators of keratinocyte activation.
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Monoclonal Antibody Register

A Set of Monoclonal Antibodies Specific for Bovine Cell Surface Molecule CD9

J. TOMÁŠKOVÁ, R. DUŠINSKÝ, L'. HOROVSKÁ, M. SIMON

CD9 antigen, a member of the transmembrane 4 superfamily (Hořejší and Vlček, 1991), is a 24 kDa surface-membrane glycoprotein present on platelets, early B cells, activated T cells, eosinophils, as well as nonhematopoietic cells like endothelial cells, fibroblasts, or glial cells (Wright and Tomlinson, 1994). Although a function for CD9 has not been clearly identified, there are indications that this molecule may be involved in cell migration, adhesion and proliferation processes whose functions may be related to its association with VLA integrins (Ikeyama et al., 1993; Masellis-Smith and Shaw, 1994; Rubinstein et al., 1994). In accordance with a signaling function, CD9 is reported to associate with small GTP-binding proteins (Seehafer and Shaw, 1991) and platelet integrins (Indig et al., 1997). CD9 antigen is also associated with transmembrane precursor of heparin-binding EGF and upregulates the juxtacrine activity of this receptor as well as its activity as the receptor for diphteria toxin (Iwamoto et al., 1994; Higashiyama et al., 1995).
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