Electronic Library of Scientific Literature


Volume 44 / No. 6 / 1997

Differential display of RNA from tumorigenic and nontumorigenic variants of hamster cells transformed with avian sarcoma virus

V. Leksa, Č. Altaner

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia

Differential display technique was applied to study expression of RNA in tumorigenic and nontumorigenic cell variants of avian sarcoma virus transformed hamster cells. Methodical conditions were worked out, which allowed identifying a cDNA fragment of an unknown gene expressed in nontumorigenic cell variant only. Its role in tumor suppression remains to be determined.

Key words: Tumorigenic and nontumorigenic cells, differential display, RNA expression.
pp. 337-341

Treatment of rat gliomas with recombinant retrovirus harboring Herpes simplex virus thymidine kinase suicide gene

J. Hlavatý, K. Hlubinová, V. Altanerová, J. Líška, Č. Altaner

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia

The retrovirus vector containing Herpes simplex virus type 1 thymidine kinase (HSVtk) gene was constructed. The vector was transfected into the packaging cell line PG13. It was shown that individual transfected cells differ in the production of recombinant retrovirus and in their susceptibility to be killed by ganciclovir. Recombinant retrovirus with a gibbon envelope was able to transduce the HSVtk gene into rat glioma cells. In vivo studies confirmed the ability of intraperitoneal ganciclovir administration to influence subcutaneous and intracerebral tumors developed after injection of C6 rat glioma cells with subsequent injection of HSVtk retrovirus producing cells.

Key words: Gene therapy, brain tumor, herpes simplex thymidine kinase, ganciclovir.
pp. 342-347

Quantitative immunocytofluorometry - new parameters for the definition of leukemia cells

O. Babušíková, M. Glasová, J. Stašáková, J. Kusenda, E. Koníková

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia

In our study we used for definition of leukemia/lymphoma cells a new parameter which allows the enumeration of mean fluorescence intensity expressed by the number of antigen molecules per cell. Quantitative immunofluorescence using calibration microbeads was performed in 36 patients with different acute and chronic lymphoid and myeloid leukemia and in 19 healthy volunteers.
We showed that quantitative immunophenotyping allowed the definition of aberrant marker densities on neoplastic cells. We demonstrated under- and overexpression of CD8 marker in CD3/CD4/CD8 complex in T acute lymphatic leukemia and T non-Hodgkin's lymphoma and T leukemia of large granular lymphocytes as compared to normal counterparts. We pointed out that certain antigens (e. g. CD10, CD4, CD24) were expressed at different levels on different cell subsets (CD10 in early B-acute lymphatic leukemia and coexpressed in T-acute lymphatic leukemia, CD4 on T cells and monocytes, CD24 on B cells and granulocytes in chronic myeloid leukemia). We showed that quantitative immune fluorescence could provide new data contributing to a more precise definition of cell differentiation. We documented the significant difference between antigen density of early and late markers in B-cell and myeloid malignancies. Further, we demonstrated that quantitative immune phenotyping could help in determination of exact definition of pathologic clone in morphologically immature leukemia population and showed that parameters of this method are also convenient for cytoplasmic marker evaluation. In our study we were able to demonstrate that CD45 quantitative expression appeared to be a more informative parameter than its percentage of antigen-positive cells as a measure of antigen expression only and we pointed out that low and high CD45 densities enabled to differentiate between pathological clone and residual healthy population in examined sample.
We showed that quantitative immune phenotyping could be another important parameter for definition of leukemia phenotype suitable for detection of minimal residual disease.

Key words: Leukemia cells, quantitative flow cytometry, immunophenotyping, CD markers.
pp. 348-355

The relationship between argyrophilic proteins and some immunophenotypic markers in acute leukemia cells

M.Klobušická, O.Babušíková

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia

This study reports the immunophenotypic features of a series of 62 selected acute leukemia patients with increased incidence of argyrophilic proteins (AgNORs) at the time of initial diagnosis. Peripheral blood and bone marrow cells of patients with T-ALL, B-precursor ALL and AML were studied. The method of silver staining was used to determine the number of AgNORs per cell. Cell surface markers were detected by a standard immunofluorescence assay. To demonstrate the relationship between AgNOR quantity and cell proliferation, the expression of activation and proliferation antigens CD38 and CD71 was investigated. To characterize the immunophenotype and the discrete stages of differentiation, the wide panel of antibodies against lymphoid, myeloid and non-lineage specific antigens was used. The number of AgNORs at diagnosis ranged from 3.05 to 6.70. Immunophenotypic analysis showed a variation in CD38 and CD71 expression among different leukemia subtypes. CD71 antigen was more expressed in T-ALL than in B-precursor ALL or in AML. Notable was the relationship between increased AgNOR quantity and antigens that characterize the immaturity of leukemic cells. The association with CD7, CD2, CD5 (without CD3 membrane expression) and CD34 in T blasts was evident. High positivity of CD19, CD10, CD34 and HLA-DR in relation to the increased amount of AgNORs in B-lineage ALL was observed. The vast majority of AML patients with high numbers of AgNORs simultaneously expressed CD13, CD33, CD34 and HLA-DR. One third of AML cases coexpressed T cell marker CD7. In conclusion, the presence of increased numbers of AgNORs at diagnosis might reflect the dependence on an early stage of leukemia cell differentiation.

Key words: Acute leukemia, argyrophilic proteins, immunophenotypic markers, cell proliferation.
pp. 356-360

Inhibitory effect of radiosensitizer AK-2123 on experimental hepatic metastases and Ca2+ active transport

N.P. Konovalova, L.M. Volkova, L.V. Tatyanenko, R.A. Kotelnikova, T.N. Yakushchenko, T.V. Kagiya

Institute of Chemical Physics, Russian Academy of Sciences, 142 432 Chernogolovka, Moscow region, Russia;
Kinki Invention Center, Kyoto, Japan

A triazole group radiosensitizer AK-2123 is shown to inhibit considerably the growth of hepatic metastases induced by the intrasplenic injection of colon adenocarcinoma cells in syngenic mice. Even an extremely low dose of the drug exhibits the antimetastatic effect. It is shown that AK-2123 injected at therapeutic dose inhibits active transport of calcium ions by the (Ca2+-Mg2+)-dependent ATP-ase. The antimetastatic effect of AK-2123 is suggested to be related, at least partially, to the inhibition of the active calcium transport.

Key words: Hepatic metastases, antimetastatic effect, radiosensitizer, active transport, calcium ions.
pp. 361-365

Human multidrug-resistant (MRP, p190) myeloid leukemia HL-60/ADR cells in vitro: Resistance to the mevalonate pathway inhibitor lovastatin

Ľ. Hunáková, J. Sedlák, M. Šuliková, J. Chovancová, J. Duraj, B. Chorváth

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia

Mevalonate pathway inhibitor lovastatin inhibited proliferation of human multidrug-resistant promyelocytic leukemia HL-60/ADR cells in vitro, with MRP-gene coded p190 mediated drug resistance, to a markedly lesser extent than that of the parental drug sensitive HL-60 cells and also that of the other human multidrug resistant (MDR-1, P-glycoprotein) myeloid leukemia cell line HL-60/VCR. The sensitivity of the examined human leukemia cell lines to the cytostatic activity of lovastatin correlated approximately with the potential of lovastatin to induce the characteristic cell cycle alteration (i.e. the accumulation of lovastatin-treated cells in the G0/G1 phase of the cell cycle).
The P-glycoprotein positive HL-60/VCR cells and the parental drug sensitive HL-60 cells were more sensitive to this cell cycle alteration than the HL-60/ADR multidrug resistant leukemia cells with MRP drug resistance. Lovastatin (72 hours, 20 ľmol) induced apoptosis and cell necrosis in HL-60 cells, apoptosis but not cell necrosis in HL-60/VCR cells and neither apoptosis nor necrosis in HL-60/ADR cells.

Key words: Lovastatin, multidrug-resistance, human leukemia, cell line, cytotoxicity, MTT test, cell cycle analysis, MDR-1, MRP, apoptosis.
pp. 366-369

The intermediate filaments and prognostically oriented morphological classification in ductal breast carcinoma

A. Rejthar, R. Nenutil

Department of Histopathology, Faculty Hospital Bohunice, 639 00 Brno, Czech Republic

The expression of cytokeratins 7, 8, 14, 18, 19 and vimentin was examined in 100 cases of ductal invasive breast carcinomas. While the predominantly diffuse immunohistological positivity of simple epithelia cytokeratins 7 (in 93), 8 (in 100), 18 (in 100) and 19 (in 97) cases represents a constant feature of these tumors, cytokeratin 14 was detected in only 36 cases which were mostly of low grade and in a focal pattern. Vimentin positivity was found in 53 intermediate and high grade tumors and, again the pattern was also rarely diffuse. The ductal carcinomas can be grouped into four classes according to vimentin and cytokeratin 14 immunoreactivity. This grouping correlates well with tumor grade and with simple histological classification of ductal breast carcinoma, consisting of the low, intermediate and high malignancy categories, as proposed here. The types of ductal carcinomas can be sorted into prognostically different subgroups, according to ICD-O morphologic terminology and commonly adopted results of morphologic and prognostic studies.

Key words: Breast cancer, histopathology, cytokeratin, vimentin, intermediate filaments, classification, prognosis.
pp. 370-373

Searching for a functional analogy between yeast Pso4 and bacterial RecA proteins in induced mitotic recombination

V. Vlčková, M. Slaninová, M.A. Morais Jr., J.A.P. Henriques, I. Fridrichová, J. Brozmanová

Department of Genetics, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia;
Departamento de Biofisica, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;
Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, 833 19 Bratislava, Slovakia

The pso4-1 mutant of S.cerevisiae is phenotypically similar to the recA mutant of E.coli; it is sensitive to DNA cross-linking agents and defective in both recombination and mutagenesis. In this paper we have measured the effect of the recA gene expression on the frequency of mitotic crossing-over and mitotic gene conversion in response to DNA damage induced by photoactivated 8-methoxypsoralen (8-MOP + UVA), ultraviolet radiation (UV) and N-methyl-N´-nitro-N-nitrosoguanidine (MNNG). The diploid pso4-1 mutant and the repair wild type strain were transformed with the multicopy plasmid carrying the recA gene placed under the control of the ADH1 promoter. The results showed that RecA is not able to restore block in induced mitotic recombination in pso4-1 cells after DNA damaging agents used. Thus RecA protein is not able to substitute Pso4 protein in homologous mitotic recombination indicating that they have probably different functions in this process.

Key words: recA gene, pso4-1 mutant, mitotic recombination.
pp. 374-379

Measurement of DNA strand breakage and DNA repair induced with hydrogen peroxide using single cell gel electrophoresis, alkaline DNA unwinding and alkaline elution of DNA

A. Gábelová, D. Slameňová, Ľ. Ružeková, T. Farkašová, E. Horváthová

Cancer Research Institute, 833 91 Bratislava, Slovakia

Three techniques: single cell gel electrophoresis (SCGE), alkaline elution of DNA (AE), and alkaline DNA unwinding (ADU) were chosen to compare the sensitivity among these methods in detection of DNA damage and repair in human diploid VH10 cell line after short-term exposure to hydrogen peroxide. Using SCGE technique a dose-dependent increase in DNA migration was found in cells exposed to hydrogen peroxide in concentration range from 10 micromol/l to 100 micromol/l. Alkaline DNA unwinding method detected increased level of single strand breaks (ssb) in concentration range from 25 micromol/l to 100 micromol/l of H2O2, and alkaline elution of DNA estimated increased DNA elution rate from concentration 50 micromol/l of H2O2. In a time course study to evaluate the kinetics of DNA repair, both SCGE and ADU techniques showed that the repair of DNA strand breaks is very rapid; the level of ssb in treated cells has returned to near the background level within two hours. After this time damage remaining in the DNA was in the form of oxidised bases as revealed the incubation of treated cells with specific DNA repair endonuclease, formamidopyrimidine-DNA glycosylase.

Key words: Hydrogen peroxide, single cell gel electrophoresis, alkaline elution of DNA, alkaline DNA unwinding, VH10 cells.
pp. 380-388

Polarographic testing of carcinogenicity of some chemotherapeutics

A. Vachálková, L. Novotný

Cancer Research Institute, Slovak Academy of Sciences, 833 19 Bratislava, Slovakia

This work is devoted to the study of polarographic reduction of three antibiotic compounds including adriamycin, chloramphenicol and erythromycin and of a synthetic antibacterial chemotherapeutic compound - 5-nitrofurantoin. The polarographic reduction was performed in the strictly anhydrous N,N-dimethylformamide with or without alpha-lipoic acid (LA) by the means of DC polarography. The values of half-wave potentials E1/2 and parameter of potential carcinogenicity were determined for the all compounds. Adriamycin was reduced during the five-step process, other compounds were reduced in two steps. The presence of LA in a polarographic solution resulted in a new polarographic one-electron wave in the range of -1.120 V to -1.790 V vs. SCE possessing a diffuse and reversible character. Its height is linearly dependent on the LA concentration in solution. The highest parameter of potential carcinogenicity tg alpha was determined for adriamycin (0.575) which belongs among compounds classified by WHO as "probably carcinogenic to humans". The lowest determined value of parameter tg alpha belonged to 5-nitrofurantoin (0.290) which has not yet been included into the IARC classification.

Key words: Adriamycin, chloramphenicol, erythromycin, 5-nitrofurantoin, DC polarography, alpha-lipoic acid, carcinogenicity.
pp. 389-394

Plasma selenium concentration in patients with stomach and colon cancer in the Upper Silesia

M. Ścieszka, A. Danch, M. Machalski, M. Dróżdż

I Clinic of Internal Diseases, Silesian Medical Academy, 40-029 Katowice, Poland;
Department of Biochemistry and Chemistry, Silesian Medical Academy, Katowice, Poland

Plasma selenium concentration was assessed in 44 patients with cancer of the gastrointestinal tract (19 subjects with stomach cancer and 25 with colon cancer) and 25 age-matched healthy control subjects. Selenium concentration was determined by the fluorometric method. The observed plasma selenium concentrations in gastrointestinal cancer patients (37.0 ą 11.05 ng Se/ml or 38.4 ą 12.6 ng Se/ml in stomach or colon cancer patients, respectively) were significantly lower as compared to the healthy age-matched control group (51.4 ą 14.4 ng Se/ml). The diagnosed low selenium status may be considered as a high risk for cancer development.

Key words: Plasma selenium, stomach cancer, colorectal cancer, selenium deficiency.
pp. 395-397

Index of authors

pp. 399-400

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