Electronic Library of Scientific Literature


Volume 46 / No. 6 / 1999


Cells producing recombinant retrovirus with thymidine kinase gene from Herpes simplex virus suitable for human cancer gene therapy

J. Hlavatý, K. Hlubinová, J. Bies, Č. Altaner

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

Therapeutic cells producing amphotropic retrovirus, which are able to transduce in vivo thymidine kinase gene of Herpes simplex virus were prepared. Single-cell clone cells with high virus productivity (PA-317JH5cl13) were obtained by cell cloning. The cells were found free of replication competent retrovirus, they were non-tumorigenic in xenogeneic host and highly sensitive to ganciclovir treatment in vitro and in vivo. The therapeutic efficacy of PA-317JH5cl13 cells was tested in rat brain tumor model. Increase in survival in the group of treated versus untreated rats was observed. Therefore, these cells are suitable for application in human clinical trial.

Key words: Retroviral vector producing cells, gene therapy, HSVtk.
NEOPLASMA, 46, 6, 1999, 329-334

Reversal of carboplatin resistance in human laryngeal carcinoma cells

L. Vuković, M. Osmak

Department of Molecular Genetics, Ruđer Bošković Institute, 10 000 Zagreb, Croatia

The effectiveness of carboplatin in the treatment of patients with tumors is limited by drug resistance. Because of that, there is a great interest to find a way to revert the resistance and improve the success of cancer treatment. The aim of the present study was to examine five potential modulators of carboplatin resistance with different mode of action: buthionine sulfoximine, ethacrinic acid, amphotericine B, cyclosporine A and aphidicoline. The effect of these compounds on the sensitivity of human laryngeal parental (HEp2) and carboplatin-resistant (HEp7T) cells to carboplatin was examined by MTT spectrophotometric assay. The results have shown that buthionin sulfoximine and, to a lesser extent, ethacrinic acid reduced the resistance of HEp7T cells to carboplatin. Aphidicolin increased the sensitivity of both HEp2 and HEp7T cells to carboplatin, but this effect was more expressed in parental HEp2 cells. Our data suggest that human laryngeal carcinoma cells treated with clinically relevant doses of carboplatin became resistant to this drug due to multifactorial molecular mechanisms. Accordingly, the resistance to carboplatin could be reduced by different modulators.

Key words: Drug resistance, carboplatin, reversal, laryngeal carcinoma cells.
NEOPLASMA, 46, 6, 1999, 335-341

Lack of correlation between repair of DNA interstrand cross-links and differential sensitivity of G0 and proliferating CD4+ lymphocytes towards cisplatin

Z. Bartošová, I.D. Horak, M. Piršel, V.A. Bohr

Metabolism Branch, National Cancer Institute, NIH, Bethesda, USA;
Laboratory of Molecular Genetics, National Institutes on Aging, NIH, Baltimore, USA;
Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovak Republic

G0 cells in a tumor are insensitive to the chemotherapeutical agents. The nature of this resistance is not completely understood. One of the factors modulating sensitivity of cells may be DNA repair of drug induced DNA damage. In this study we have compared gene-specific formation and repair of cisplatin-induced interstrand cross-links (ICL) in human G0 and proliferating CD4+ lymphocytes. Cisplatin killing of G0 CD4+ lymphocytes is inefficient, and these cells resemble those in a tumor. After exposure to cisplatin under similar conditions, the frequency of ICL introduced is twice as high in the proliferating compared to the resting lymphocytes. Repair of ICL was measured in the housekeeping gene, dihydrofolate reductase (DHFR), in the proliferation inducible c-myc gene, and in the inactive delta-globin gene. We observed similar relative rates and extent of ICL repair in all three genes studied, in G0 or proliferating CD4+ lymphocytes. The mechanisms responsible for the resistance of G0 CD4+ lymphocytes towards cisplatin are discussed.

Key words: Cisplatin, interstrand cross-link, gene-specific DNA repair, G0 and proliferating lymphocytes.
NEOPLASMA, 46, 6, 1999, 342-348

Flow cytometric analysis of some activation/proliferation markers on human thymocytes and their correlation with cell proliferation

O. Babušíková, V. Ondráčková, J. Prachař, J. Kusenda, V. Hraška

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovak Republic;
Children?s Cardiocenter of University Children´s Hospital, 833 40 Bratislava, Slovak Republic

We immunophenotyped cells from ten human thymuses with emphasis on expression of the CD38 and CD71 antigens. These antigens play role in activation cells and increased expression of them was observed in some leukemia. Simultaneously, certain attention has also been devoted to some further activation markers, e.g. CD25, CD26 and HLA-DR. The classification of leukemia is based on comparison of normal and pathological cells. The study of expression of CD38, CD71 and other markers on thymocytes simultaneously with DNA analysis can be useful for answer if expression of CD38 and CD71 on pathologic cells is a sign of their proliferative ability, a part of immature phenotype in some leukemia, or it is a case of aberrant immunophenotype. In our study, 94% thymocytes were CD38+ and only 16% were CD71+. From our immunophenotypic results including MESF (molecules of equivalent soluble fluorochrome) values and analysis of the cell cycle, the conclusion could be drawn that antigen CD71 can participate in regulation of thymocyte development and presence of both - CD38 and CD71 on pathologic cells will be in all probability the case of aberrant phenotype. We observed a clear correlation of the percentage and MESF values of CD71-positive cells with the cell proliferation only after in vitro thymocytes stimulation with PHA and IL-2. In summary, a strong parallelism was observed regarding the positive relationship between the proliferative rate (assessed by the number of S-phase cells) of stimulated thymocytes and the quantitative (% and MESF) values of some markers - CD71, CD25, CD26 and HLA-DR and negative one with CD38 marker values.

Key words: Thymocytes, immunophenotyping, MESF values, DNA analysis, CD38 and CD71 markers, flow cytometry.
NEOPLASMA, 46, 6, 1999, 349-355

Reduction of genotoxic effects of MNNG by butylated hydroxyanisole

E. Horváthová, D. Slameňová, S. Bonatti, A. Abbondandolo

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovak Republic;
Laboratory of Mutagenesis, National Institute for Cancer Research, Genova, Italy

Butylated hydroxyanisole (BHA) is a food preservative with markedly contradictory effects. On one side many studies showed its antimutagenic and anticarcinogenic effects but on the other side dietary levels of BHA were reported to cause gastrointestinal hyperplasia in rodents. We studied the influence of BHA on cytotoxicity, mutagenicity, and DNA-damaging activity of N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) in Chinese hamster V79 cells cultured in vitro. Our results showed that BHA significantly reduced the frequency of 6-thioguanine resistant (6-TGr) mutations and micronuclei induced in V79 cells by MNNG. These antimutagenic effects of BHA were, however, accompanied by a very marked increase of MNNG toxicity and also slightly increased level of MNNG-induced DNA damage.
For evaluation of toxicity we used three methods: (i) trypane blue exclusion; (ii) plating efficiency; and (iii) intensity of cellular macromolecule synthesis. The level of DNA damage was measured by the comet assay. On the basis of obtained results we suggest that BHA, which induces phase II detoxifying enzymes, probably doesn?t reduce the level of DNA damage induced in time of MNNG-treatment but it reduces the level of DNA damage created during a long-term period needed for expression of 6-TGr mutations and micronuclei.

Key words: Chinese hamster cells V79, butylated hydroxyanisole, N-methyl-N´-nitro-N-nitrosoguanidine, mutagenicity, cytotoxicity.
NEOPLASMA, 46, 6, 1999, 356-362

7-Ethoxyresorufin O-deethylase (EROD) activity is not capable of reflecting the overall malignant potential of breast cancer tissue

M. Iscan, B.C. Eke, S. Aygörmez, T.Çoban, D. Bülbül

Department of Toxicology, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey;
Department of Pathology, Demetevler Oncology Hospital, Ankara, Turkey

7-Ethoxyresorufin O-deethylase (EROD) (mainly catalyzed by cytochrome P450 (CYP) 1A1 and used as a marker for CYP1A1) activity was measured in the breast tumor and surrounding tumor free (normal) tissues of 37 female breast cancer patients with infiltrating ductal carcinoma. About 11% of the tumor and normal breast tissue samples lacked the enzyme activity. Large interindividual variations in the activities of EROD were found in both tumor and normal tissues ranging from 0 to 283 and 0 to 801 fmol/mg/min, respectively. However, no significant difference was noted between the mean EROD activities of tumor and normal breast tissues. This tendency did not change with the stage and grade of the malignancy and menopausal status. No significant correlation was observed between the EROD activity and stage or grade of malignancy (p > 0.05). Thus, it appears that EROD activity is not capable of reflecting the overall malignant potential of breast cancer tissue.

Key words: Human, breast cancer tissue, 7-ethoxyresorufin O-deethylase.
NEOPLASMA, 46, 6, 1999, 363-367

Flow cytometry of p53 protein expression in some hematological malignancies

E. Koníková, J. Kusenda, O. Babušíková

Cancer Research Institute, Slovak Academy of Sciences, 83 391 Bratislava, Slovak Republic

p53 is a tumor suppressor gene encoding a nuclear phosphoprotein that plays an important role in the control of normal cell proliferation. We have tried to establish the value of the p53 protein expression in peripheral blood (PB) and/or bone marrow (BM) cells of patients with some hematological malignancies. A recently developed fixation/permeabilization method was modified for flow cytometric assessment of p53 protein expression using two anti-p53 monoclonal antibodies. p53 quantitation expressed as molecules of equivalent soluble fluorochrome per cell (MESF) providing valuable data contributing to a more precise definition of leukemic cells, was also applied.
Our findings showed higher percentage of p53 expression in cells of AML patients at the time of diagnosis opposite to the controls. These data, in association with immunophenotype of cells, accompanied diagnosis of relapse or definition of remission after allogeneic BM transplantation. We observed also elevated levels of p53 protein at initial diagnosis of early B-ALL. According to our results quantitation of p53 protein allows better characterization of selected population of BM cells and should be used for the monitoring of blast persistence during and after therapy and might also be one of the methods to indicate early relapse. Percentage of p53 protein positivity varied in our group of B-CLL patients tested in connection with progression of disease. We documented also one case of Burkitt´s lymphoma with high percentage of p53 positivity.
Measurement of p53 protein expression by flow cytometry may be of clinical importance by indicating levels of positivity. Our results suggest, that p53 alteration is frequently involved at initial diagnosis of AML, in some T-cell disorders and on the contrary more frequently during early B-ALL relapse, in advanced stages of B-CLL and in Burkitt´s lymphoma. p53 protein quantitation is of value to ascertain malignancy and provides additional parameter suitable for the evaluation of residual disease and for the monitoring of therapy.

Key words: p53, flow cytometry, quantification, fixation/permeabilization, leukemia cells.
NEOPLASMA, 46, 6, 1999, 367-376

Incidence of micronuclei in cytokinesis-blocked lymphocytes of medical personnel occupationally exposed to ultrasound

V. Garaj-Vrhovac, N. Kopjar

Institute for Medical Research and Occupational Health, HR-10001 Zagreb, Croatia

In order to investigate possible DNA damaging effects of ultrasound, the micronucleus assay on cytokinesis blocked human lymphocytes was performed. Preparations were stained by conventional Giemsa staining technique combined with additional staining techniques using fluorescent dye DAPI and silver nitrate. Blood samples were taken from medical personnel employed on ultrasonic scanning in medical diagnosis and unexposed control subjects from general population. Lymphocytes were cultivated in vitro at 37oC. Cytochalasin-B in final concentration of 6 microg/ml was added 44 h after mitogen stimulation and cultures were harvested 28 h thereafter. Staining with both additional techniques can be used to distinguish micronuclei originating from breakage or mitotic loss of certain human chromosomes bearing DAPI-positively stained or silver-positively stained regions. The results obtained indicate statistically significant increases in total number of micronuclei and changes in their distribution in exposed subjects compared to control. Based on different intensity of DAPI staining signal-positive and signal-negative ?type? of micronuclei are distinguished, while silver staining has revealed Ag-NOR+ and Ag-NOR- micronuclei. In exposed subjects a prevalence in number of Ag-NOR+ micronuclei over Ag-NOR- micronuclei compared to control was observed, indicating greater susceptibility of chromosomes from D and G groups to damage caused by continuous occupationally exposure to ultrasound. In spite of their limitations, our results indicate that combination of conventional Giemsa staining of micronuclei with fluorescent dye DAPI and silver nitrate staining techniques can be valuable complement to the standard micronucleus assay.

Key words: Ultrasound, human lymphocytes, micronuclei, Giemsa, DAPI, silver staining.
NEOPLASMA, 46, 6, 1999, 377-383

p53 status in breast carcinomas revealed by FASAY correlates well with p53 protein accumulation determined by immunohistochemistry

J. Šmardová, V. Vagunda, E. Jandáková, M. Vagundová, H. Koukalová, J. Kovařík, J. Žaloudík

Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic;
University Hospital Bohunice, Brno, Czech Republic

The prognostic and predictive value of p53 mutation in breast cancer is still conflicting. The choice of the p53 status detection method may account for some discrepancies. In this pilot study we compared two differently-based methods for detection of p53 alteration in 32 breast carcinoma samples: the immunohistochemical method using Bp53, DO1 and DO11 monoclonal antibodies for analysis of the p53 protein accumulation in cell nuclei and the functional method FASAY. FASAY - functional analysis of the separated alleles in yeast - tests the capability of the human p53 to transactivate a reporter with a p53 binding site RGC driving the ADE2 gene in yeast. In our group the percentage of breast cancers with accumulated p53 protein was 50%, as well as percentage of mutant p53 scored by FASAY was 50%. Although the agreement of both methods, when comparing the results of individual patients was high (94%), our results show that immunohistochemistry does not reflect the p53 status quite exactly.

Key words: Breast cancer, p53, FASAY, immuno-histochemistry.
NEOPLASMA, 46, 6, 1999, 384-389

Heterogeneity of keratin intermediate filaments expression in human glioma cell lines

I. Máčiková, A. Peržeľová, P. Mráz, J. Šteňo, I. Bízik

Department of Anatomy, School of Medicine, Comenius University, 813 72 Bratislava, Slovak Republic;
Department of Neurosurgery, Dérer?s Hospital, Bratislava, Slovak Republic

Keratin intermediate filaments (Ifs) are specific for epithelial cell differentiation. This study demonstrates the presence of keratin in two recently established human glioblastoma cell lines 8-MG-BA and 42-MG-BA. Immunofluorescence staining was performed on cells within passage 230 to 235 using monoclonal pan-cytokeratin antibodies. The cells were analyzed during several DIV at different cell density. Keratin-positive stained cells reached 5 to 7% in 8-MG-BA and less than 0.1% in 42-MG-BA cell line. The presence of keratin-positive cells was independent on cell density and days in vitro. Keratin-positive cells appeared unevenly distributed in both cell lines. They were observed as single or areas of keratin-positive cells. The morphological features of keratin-positive and keratin-negative cells were similar. The results are discussed with respect to previous studies on glial fibrillary acidic protein (GFAP) and vimentin to show the heterogeneity of IFs expression in glioma cell lines.

Key words: Intermediate filaments, keratin, GFAP, vimentin, glioma cell lines, brain tumors.
NEOPLASMA, 46, 6, 1999, 390-393

Comparison of two non-anthracycline-containing regimens for elderly patients with diffuse large-cell non Hodgkin?s lymphoma - possible pitfalls in results reporting and interpretation

S. Jelić, N. Milanović, Z. Tomašević, S. Matković, D. Gavrilović

Institute of Oncology and Radiology in Serbia, 11 000 Belgrade, Yugoslavia

Age over 65 years is a risk factor per se for doxorubicin administration, and coexisting diseases pose additional problems. There is still controversy whether chemotherapy regimens for elderly patients with aggressive NHL should be full-dose doxorubicin containing or whether development of non-anthracycline containing regimens is warranted.
In this prospective study, 47 patients aged over 65 years with diffuse large cell NHL clinical Stage I/IE bulky-IV and no other initial exclusion criteria were randomized to receive either BCNU 120 mg/m2 d. 1, VP16 60 mg/m2 d. 2-4, procarbazine 85 mg/m2 d. 2-8 (arm A, 27 patients) or mitoxantrone 6 mg/m2 d. 1. with VP16 and procarbazine in the same dosage and schedule (Arm B, 20 patients). Partial responders received additional irradiation treatment if feasible. Arms were well balanced according to age, sex, clinical stage and performance status. Ten patients from arm A and 13 from arm B had PS 2 or 3; 14 patients from arm A and 8 from arm B had clinically significant antecedent and/or concomitant disease (SACD: cardiac, vascular, cerebrovascular, neurological, renal or other). On the intent-to-treat basis, the results were the following. Arm A: median number of cycles 3 (range 1-6); early death 3 patients; 16/27 responses (59%), 7 complete (30%). Arm B: median number of cycles 3 (range 1-6); early death 4 patients; 12/20 responses (60%), 3 complete (15%). There was no difference either in response rate or survival between the two arms, and pooled results from the two arms displayed a plateau on the survival curve from the 20-th month onwards on the probability level of 0.40. Clinical stage of NHL, bulky disease, age and sex did not influence survival. Initial performance status did influence survival at the significance level of p = 0.045. Although presence of SACD did not influence initial performance status, it had a strong negative impact on survival (p = 0.0004).
The results point to the existence of two prognostic categories of elderly patients with large cell NHL, one with a poor survival, the other achieving a significant response rate and relapse free survival. Comorbidity (SACD) apparently accounts for the poor survival in a subpopulation of elderly patients. Clinical trials with elderly patients with NHL with PS 0 or 1 and no serious coexisting disease as inclusion criteria, analyzed on an evaluable patients basis, target only to a prognostically better subpopulation among these patients.

Key words: Non Hodgkin?s lymphoma, chemotherapy, elderly, prognostic factors, comorbidity.
NEOPLASMA, 46, 6, 1999, 394-399

Short communications

NEOPLASMA, 46, 6, 1999, 400-404