Electronic Library of Scientific Literature


Volume 45 / No. 4 / 1998

Increasing DNA repair capacity in bone marrow by gene transfer as a prospective tool in cancer therapy

K. Kleibl, G.P. Margison

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia;
CRC Section of Genome Damage and Repair, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 9BX, U.K.

Resistance of tumor cells to alkylating anticancer agents that produce adducts at the O6 position of guanine in DNA, the O6-alkylating agents, correlates with the expression of O6-alkylguanine-DNA alkyltransferase (ATase). O6-benzylguanine and related pseudosubstrates are able to inactivate human ATase in vitro and in vivo and they are being tested as chemotherapeutic adjuvants for enhancing the effectiveness of O6-alkylating drugs. On the other hand, the clinical consequences of ATase depletion may be fatal for some sensitive systems e.g. hematopoiesis. To overcome this problem, strategies for the protection of primary bone marrow cells by targeted transfer of pseudosubstrate-resistant ATase genes have been considered and recently achieved at the laboratory level. This approach could therefore be now extended to a clinical cancer gene therapy program.

Key words: Alkylating agents, chemotherapy, O6-alkylguanine-DNA alkyltransferase, nitrosourea, DNA repair inhibition, hemopoetic stem cells.
pp. 181-186

Application of NMR spectroscopy in biochemical studies of tumor cells sensitive and resistant to anticancer drugs

E. Hanušovská, I. Dovinová, I. Tkáč, L. Novotný

Cancer Research Institute, Slovak Academy of Sciences, SK-833 91 Bratislava, Slovakia;
Center of Magnetic Resonance Research, University of Minnesota, Medical School, Minneapolis, USA;
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, Kuwait

Drug resistance is a prominent problem of cancer therapy. Differences in quantity and quality of many metabolites in normal and malignant cells and their changes after treatment by anticancer drugs can be detected by nuclear magnetic resonance (NMR) both in vivo and in vitro. The results of in vivo and in vitro 1H, 13C, 19F and 31P NMR spectroscopy and their correlation with the degree of resistance to anticancer drugs are discussed. Monitoring of treatment and development of drug resistance by this non-invasive method could be useful not only in cancer research related to drug resistance but also in clinical medical oncology.

Key words: Cancer therapy, drug resistance, in vivo and in vitro NMR,1H,13C,19F and 31P NMR spectroscopy.
pp. 187-197

Expression of cytokine receptors on different myeloid leukemic cells

K. Koubek, A. Kumberová, J. Starý, O. Babušíková, H. Klamová, M. Filipec

Institute of Hematology and Blood Transfusion, 128 20 Prague 2, Czech Republic;
2nd Pediatric Department, Faculty Hospital, Prague, Czech Republic;
Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia;
2nd Department of Ophtalmology, 1st Medical Faculty of Charles University, Prague, Czech Republic

We have studied the expression of cytokine receptors CD25 (IL-2Ra,55kD), CD116 (hGM-CSRF,145kD), CD117 (CSFR,145kD), CD120a (TNFR,55kD), CD120b (TNFR,75kD), CD121a (IL-1R, type I,80kD), CD123 (IL-3R), CD124 (IL-4R,140kD), CD126 (IL-6R, 80kD), CDw127 (IL-7R, 75kD), CDw128 (IL-8R), CD130 (gp130 subunit), CD131 (common beta), CD134 (OX40) and also CD95 (Fas antigen) on the myeloid leukemic cells. Cells from peripheral blood or bone marrow of 30 patients with disorders in myeloid lineage included mostly acute myeloid leukemias (with high leukocyte count and percentage of blasts) were analyzed for the expression of surface membrane molecules by indirect immunofluorescence method evaluated by flow cytometry. The findings indicate that some monoclonal antibodies have a reactivity against cytokine receptors of pathological cells in individual cases, but with very variable qualitative and quantitative (number copies/cell) expression (preliminary results). The leukemic cells demonstrate unique cytokine receptor profiles, which reveal the great diversity of immunophenotypes within the main functional characterization of blood malignancies. The immunophenotype heterogeneity of leukemic cells has proved to be much greater than to match with existing classification criteria. This fact could raise the necessity of further evaluation and specification of cytokine markers of the myeloid acute leukemias. On the other hand, detection of cytokine receptors on the leukemia cells is important for cytokine therapy.

Key words: Blood malignancies, myeloid leukemic cells, receptors for growth factors, immunofluorescence analysis.
pp. 198-203

Staurosporine enhanced benzamide riboside-induced apoptosis in human multidrug-resistant promyelocytic leukemia cells (HL-60/VCR) in vitro

ź. Hunáková, J. Duraj, D. Romanová, L. Novotný, J. Sedlák, M.R. Kelley, T. Szekeres, H.N. Jayaram, B. Chorváth

Department of Molecular Immunology, Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia;
Department of Experimental Therapy, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia;
Clinical Institute for Medical and Chemical Laboratory Diagnostics, General Hospital of Vienna, University of Vienna Medical School, Vienna, Austria;
Indiana University School of Medicine, Indianopolis, USA

The inosine monophosphate (IMP) dehydrogenase inhibitor benzamide riboside (BR) induced apoptosis (detected with the aid of flow cytometric identification of cells with sub-G0 DNA content and increased side angle light scatter) equally or slightly more intensively in the multidrug-resistant human promyelocytic leukemia cell line (HL-60/VCR: MDR-1 gene, Pgp positive) in comparison with the parental drug sensitive HL-60 cells. Staurosporine alone induced relatively low level of apoptosis in parental HL-60 cells but higher level (approximately 35%) of apoptosis in multidrug-resistant HL-60/VCR cells after 24 hour induction. The combination of benzamide riboside and staurosporine induced in both drug-sensitive and drug-resistant HL-60 cells a marked proportion of apoptotic cells already after short (6 hour) induction (more than 30% of apoptotic cells).

Key words: Benzamide riboside, staurosporine, apoptosis, flow cytometry, human leukemia cells, drug-resistant, MDR-1, P-glycoprotein.
pp. 204-209

The pathomorphology of a human xenotransplanted basaloid squamous cell carcinoma

I. Babó, A. Zalatnai, Zs. Schaff, Zs. Suba, Gy. Szabó, A. Jeney

Ist. Institute of Pathology and Experimental Cancer Research, Semmelweis University of Medicine, H-1085 Budapest, Hungary;
Department of Oral- and Maxillofacial Surgery, Semmelweis University of Medicine, Budapest, Hungary

To elucidate some factors related to the malignant phenotype of an oral tumor with mixed cell population the question has been raised whether the biological behavior of the basaloid or the squamous cells show any difference in an immunosuppressed host organism.
Basaloid squamous cell carcinoma (BSCC) surgically removed from sublingual location was xenotransplanted either subcutaneously or in the oral submucosa and the histology, ultrastructures, LDH isoenzyme pattern were investigated.
The epithelial origin of the established tumor line (HTB-1) could be recognized according to the characteristic epithelial ultrastructures, while the type of the LDH isoenzymes proved its human origin. The squamous cell population dominating the parent surgical specimen of BSCC regressed during xenotransplantation in the subcutan location, on the contrary the basaloid cells grew and maintained the tumor. Interestingly the basaloid cells transplanted from the subcutis to the oral submucosa generated a squamous cell population with an infiltrative growth pattern.
The xenografted BSCC offer a promising model to investigate the contribution of each cell populations in the malignant phenotype.The presented data indicate that the basaloid cells are responsible for maintaining the tumor cell population, but certain malignant features (i.e. infiltrative growth) is associated to the squamous cells which are generated from the basaloid cells only under specific circumstances. Thus this particular model system showed that different malignant features could be associated to the basaloid and to the squamous cell component.

Key words: Basaloid, squamous cell carcinoma, xenograft, orthotopic transplantation, invasive growth.
pp. 210-215

A 65 kDa oncofetal protein (p65), proliferating cell nuclear antigen (PCNA) and Ki67 expression in breast cancer patients

H. Niewiadomska, M. Mirowski, M. Stempien, B. Olborski, J.Z. Blonski, M. Hanausek, R. Wierzbicki

Department of Oncology, Medical University, Lodz, Poland;
Department of Surgery, Oncological Center of Lodz, Poland;
Department of Hematology, Medical University of Lodz, Poland;
The University of Texas MD Anderson Cancer Center, Science Park Research Division, USA;
Department of Biochemistry, Institute of Environmental Research and Bioanalysis, Medical University, 90-151 Lodz, Poland;
Radioisotope Center Polatom Otwock-Œwierk, Poland

Paraffin-embedded tissue slides from 89 infiltrating ductal breast carcinoma, 10 fibrocystic disease and 10 fibroadenoma were assessed immunohistochemically using monoclonal antibodies against human p65 antigen and polyclonal antibodies against p65-like protein present in fetal bovine serum. We did not find any evident differences in p65 detection by polyclonal and monoclonal antibodies, however, monoclonal antibody seems to be more specific. This factor is not induced by cellular proliferation associated with nonneoplastic diseases what was confirmed by immunohistochemical analysis of expression of p65 protein and well know markers of proliferation (proliferating cell nuclear antigen - PCNA and Ki 67). It was established that there is no correlation between p65 and PCNA or Ki67 expression. High proliferating indexes (PI) for PCNA (PI-PCNA) or Ki67 (PI-Ki67) may help in selection of tumors with high proliferating activity independently from histological grade of malignancy established by routine methods. The estimation of p65 protein may be useful in the selection of precancerous changes and more differentiated ductal cancer of the breast what raises the possibility that p65 antigen may be helpful in the screening examination of women with high risk for cancer development.

Key words: Oncofetal protein, anti-p65 antibody, proliferating cell nuclear antigen (PCNA), Ki 67, breast cancer.
pp. 216-222

Inhibition of apoptosis is the cause of resistance to doxorubicin in human breast adenocarcinoma cells

M. Osmak, A. Brozović, A. Ambriović-Ristov, M. Hadžija, B. Pivčević, T. Smital

Department of Molecular Genetics, Ruđer Bošković Institute, 10000 Zagreb, Croatia;
Department of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia;
Center for Marine Research, Ruđer Bošković Institute, Zagreb, Croatia

In our previous paper we have described the isolation and characterization of a doxorubicin (DOX) resistant subline of breast adenocarcinoma SC6 cells. These cells were obtained after the treatment with low, clinically relevant doses of doxorubicin. They became cross-resistant to different wide by used cytostatics. The expression of several genes involved in mitotic signal transduction, as well as cathepsins D and L, was similar in both parental and doxorubicin treated cells. The aim of this study was to examine the molecular mechanisms involved in resistance of these cells to doxorubicin.
Activity of plasma membrane Pgp was examined in parental and resistant cells due to rhodamine-accumulation assay. The involvement of glutathione (GSH) and glutathione S-transferase (GST) in resistance to doxorubicin was determined in MTT modified assay due to the addition of specific inhibitors: buthionine sulfoximine (for GSH) or ethacrynic acid (for GST). The kinetic of apoptosis was followed after the treatment with DOX in control and SC6 cells by fluorescent microscope. The occurrence of apoptosis was confirmed by analysing DNA fragmentation in agarose gel.
Our results indicate that P-glycoprotein, glutathione or glutathione transferases were not involved in resistance of SC6 cells to doxorubicin. However, the apoptosis was inhibited in doxorubicin-resistant cells. Therefore, even low doses of doxorubicin can induce the resistance to this drug due to inhibition of apoptosis.

Key words: Drug resistance, doxorubicin, breast adenocarcinoma cells, apoptosis.
pp. 223-230

Expression of CD10, CD19 and CD34 markers in bone marrow samples of children with precursor B-cell acute lymphoblastic leukemia in clinical and hematological remission

J. Čáp, O. Babušíková, E. Kaiserová, M. Jamárik

Department of Childhood Oncology, University Pediatric Hospital, 833 40 Bratislava, Slovakia;
Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia

Long-term follow-up of the rate of CD10, CD19 and CD34 antigens expression compared with the percentage of lymphocytes and blastic cells in 196 bone marrow specimens of 91 children with early B-cell acute lymphoblastic leukemia has been performed. It was shown that during a five year period there were no significant differences concerning the percentage of CD markers and the percentage of lymphocytes and blastic cells except those in the 4th year of clinical and immunological follow-up - when increase of lymphocytes and CD19 marker percentage have been observed. Consensus, i.e. more than 20% CD markers positivity associated with more than 5% blasts (positive consensus) or less than 20% CD markers positivity combined with less than 5% blasts (negative consensus) was ascertained in 92.8% of cases. Disagreement, i.e. the rate of CD markers over 20% combined with less than 5% blasts was found in 14 (7.2%) patients.This finding present in the initial phase of the disease could be considered as a sign of not complete remission, in contrast to the finding in children who completed chemotherapy and were in long-term hematological remission, in which the increase of CD markers could be considered as a sign of increased regeneration activity of bone marrow after chemotherapy induced medullary aplasia. Anyway, the increase of CD10, CD19 and CD34 antigens in bone marrow samples over 20% should be evaluated with caution. In these cases, mainly in children with long-term hematologic remission the examination should be repeatedly performed and/or more sensitive methods for detection of minimal residual disease should be applied. In control group of 20 children without leukemia the rate of CD markers in the bone marrow was always under 20%.

Key words: Early B-cell ALL in children, immunophenotyping, flow cytometry, CD10, CD19, CD34 markers, clinical and hematological remission.
pp. 231-236

Immunophenotypic characteristics of T-acute lymphoblastic leukemia cells in relation to DPP IV enzyme expression

M. Klobušická, O. Babušíková

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

In the present study we have examined immunophenotypic characteristics of T-acute lymphoblastic leukemia (T-ALL) cells in relation to the expression of enzyme dipeptidyl peptidase IV (DPP IV). Peripheral blood and bone marrow cells of T-ALL patients at diagnosis were estimated. Cell surface markers were detected by a standard immunofluorescence assay and FACStar flow cytometry using a broad panel of monoclonal antibodies to define T-cell immunophenotype. DPP IV activity was investigated in phenotypically defined T-lymphoblasts. Association between DPP IV expression and proliferation was monitored by the expression of CD71 and CD38, which could be considered as markers of activation and proliferation, and by the silver-staining of nucleolar organizer regions-related proteins (argyrophilic proteins). Lymphoblasts, divided according to the presence or absence of DPP IV activity revealed remarkable heterogeneity in the immunophenotypic features. The vast majority of DPP IV positive T-ALL cases expressed CD4, CD8, CD7, CD5, CD2 along with CD71 and CD38 antigens, but the cells were surface membrane CD3 antigen negative. The phenotype of DPP IV negative cases displayed membrane CD3 antigen and variable expression of CD4 and CD8. CD71 and CD38 were frequently negative. It appears, that DPP IV active cells form the population with immature phenotype, as evidenced by mCD3 antigen absence. Relation between DPP IV positive cells and proliferation activity of T-blasts was observed, given by the presence of CD71 and CD38 positivity and overexpression of argyrophilic proteins (AgNORs). In conclusion, our study indicates a close relationship between DPP IV activity and the features of T-cell immaturity. Association among DPP IV, CD71, CD38 and AgNORs might reflect possible relationship between immature phenotype and proliferative ability of blast cells in T-ALL patients.

Key words: T-acute lymphoblastic leukemia, immunophenotype, DPP IV, cell proliferation, argyrophilic proteins.
pp. 237-242

Evaluation of potential carcinogenicity of steroidal alkaloids from Veratrum album L. by the DC polarography method

A. Vachálková, D. Grančai, M. Nagy, L. Novotný

Department of Experimental Therapy, Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia;
Department of Pharmacognosy and Botany, Faculty of Pharmacy, Comenius University, Bratislava, Slovakia;
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, Kuwait

The presented work is devoted to the study of polarographic reduction in the series of 13 alkaloids isolated from various parts of Veratrum album subsp. lobelianum. The used compounds were evaluated from the point of view of their potential carcinogenicity in anhydrous N,N-dimethylformamide (DMF) by the method of DC polarography. All compounds were reduced during an one two-electron irreversible step. Their potential carcinogenicity characterized by a parameter tg alpha value determined in the presence of alpha-lipoic acid ranged from the highest value 0.257 obtained for the solanidane skeleton containing rubijervine to the value 0.070 for jervine. The tg alpha value determined for rubijervine (0.257) is comparable with the tg alpha of naphto-(2',1',2,3)fluoranthene (0.270)-compound classified by IARC as possible carcinogen for human. The tg alpha values determined for other alkaloids were relatively low and they do not indicate any possible carcinogenic activity.

Key words: Veratrum alkaloids, DC polarography, carcinogenic activity.
pp. 243-247

Direct interaction between verapamil and doxorubicin causes the lack of reversal effect of verapamil on P-glycoprotein mediated resistance to doxorubicin in vitro using L1210/VCR cells

A. Breier, Z. Drobná, M. Barančík

Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava, Slovakia;
Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovakia

Mouse leukemic cell subline L1210/VCR exerts expressive multidrug resistance (MDR) that is mediated by P-glycoprotein. Cells originally adapted to vincristine are also extremely resistant to doxorubicin. Resistance to both vincristine and doxorubicin is connected with depression of drug uptake. While resistance of L1210 cells to vincristine could be reversed by verapamil as chemosensitizer, resistance of cells to doxorubicin was insensitive to verapamil. Action of verapamil (well-known inhibitor of PGP activity) on multidrug resistance was often used as evidence that MDR is mediated by PGP. From this point it may be possible that the resistance of L1210/VCR cells to vincristine is mediated by PGP and the resistance to doxorubicin is mediated by other PGP-independent system. Another and more probable explanation of different effect of verapamil on resistance of L1210/VCR cells to vincristine and doxorubicin may be deduced from the following fact: Using UV spectroscopy we found that doxorubicin dissolved in water buffered medium interacts effectively with verapamil. This interaction may be responsible for the decrease of concentration of both drugs in free effective form and consequently for higher survival of cells. In contrast to doxorubicin vincristine does not give any interaction with verapamil that is measurable by UV spectroscopy and resistance of L1210/VCR cells to vincristine may be fully reversed by verapamil.

Key words: Multidrug resistance, P-glycoprotein, vincristine, doxorubicin, verapamil.
pp. 248-253

Macrocyclic Cu(II)tetraanhydroaminobenzaldehyde complex - antiproliferative activity in vitro and in vivo

S. Jantová, Z. Ďuračková, L. Novotný, M. Urbančíková, I. Dovinová, J. Labuda, L. Wsolová

Department of Biochemistry and Microbiology, Faculty of Chemical Technology, Slovak University of Technology, 812 37 Bratislava, Slovakia;
Department of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, Comenius University, Bratislava, Slovakia;
Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia;
Department of Comparative Toxicology, Institute of Preventive and Clinical Medicine, Bratislava, Slovakia;
Department of Analytical Chemistry, Faculty of Chemical Technology, Slovak University of Technology, Bratislava, Slovakia;
Department of Epidemiology and Biometrics, Institute of Preventive and Clinical Medicine, Bratislava, Slovakia

The macrocyclic Cu(II)-tetraanhydroaminobenzaldehyde complex Cu(TAAB)Cl2 induces various cytotoxic effects in the dependence on a cell line, concentration and time of exposition.
The highest complex concentration of 914 nmol induces degeneration of certain part of the HeLa cells population after 24 hours of cultivation. The concentration of 183 nmol causes a delayed cytotoxic effect on HeLa and HepG2 cells. After 24 hours of culturing 15.4-28.4% of cell population proliferated but after 48 and 72 hours 2.0-42.3% of the cell population degenerated.
The cytotoxic effect on V79 cells is directly dependent on the actual concentration and time of the complex influence. The cytotoxic concentrations of Cu(TAAB)Cl2 induce an integrity damage of cytoplasmatic membrane and two phase unbalanced growth. Cu(TAAB)Cl2 possesses an antileukemic activity which at the dose of 10 mg/kg of body weight is not accompanied by side toxic effects on mice.

Key words: In vitro interaction, in vivo interaction, Cu(II)-tetraanhydroaminobenzaldehyde complex, unbalanced growth, integrity of cytoplasmatic membrane.
pp. 254-260

Dyserythropoietic changes and sideroblastic anemia in patients with hairy cell leukemia before and after therapy with 2-chlorodeoxyadenosine

P. Žák, L. Chrobák, K. Podzimek, L. Plíšková, K. Dľdič

Department of Clinical Hematology, Teaching Hospital, Charles University, Faculty of Medicine, 500 05 Hradec Králové, Czech Republic;
Institute of Clinical Biochemistry and Diagnostics, Teaching Hospital, Charles University, Faculty of Medicine, Hradec Králové, Czech Republic;
Institute of Pathology, Teaching Hospital, Charles University, Faculty of Medicine, Hradec Králové, Czech Republic

Active hairy cell leukemia is associated with an increase of RDW (Red Cell Distribution Width) which normalizes after successful therapy with 2-chlorodeoxyadenosine (2-CdA) [9]. To clarify this phenomenon, bone marrow films performed before therapy with 2-CdA and after its successful completion were subjected to careful evaluation. Dyserythropoietic changes were present in 5 out of 17 patients before the therapy with 2-CdA. In 2 patients the changes were only slight, characterized by irregularities of the shape of nucleus and nuclear contour, in the remaining 3 patients the changes were marked, represented by nuclear lobulation, karyorrhexis and binuclearity, with the presence of ringed sideroblasts in one of them. After therapy with 2-CdA complete hematologic remission was achieved in 4 patients with disappearance of dyserythropoietic changes and normalization of RDW values. In the last patient with ringed sideroblasts despite complete remission with disappearance of tumoral cells in the bone marrow as demonstrated by immunohistochemical analysis of trephine bone marrow biopsy with monoclonal antibody DBA 44 the condition deteriorated, RDW remained unchanged, the sideroblastic anemia progressed.

Key words: Hairy cell leukemia, dyserythropoiesis, sideroblastic anemia.
pp. 261-265

The influence of rutin on the weight, metastasis and melanin content of B16 melanotic melanoma in C57BL/6 mice

G. Drewa, D.O. Schachtschabel, K. Pałgan, A. Grzanka, R. Sujkowska

Department of Human Biology, University School of Medical Science, 85-092 Bydgoszcz, Poland;
Department of Physiologic Chemistry, Philipps University, Marburg, FRG;
Department of Pathomorphology, University School of Medical Science, Bydgoszcz, Poland

The influence of rutin on the growth rate and tumor weight of B16 melanoma as well as melanin content and ultrastructure of melanoma cells and metastasis formation was studied in mice.
All mice were injected s.c. into the left flank with 0.2 ml of suspension containing 106 B16 melanoma cells. The experimental groups were treated with a solution of rutin i.p. every two days with total doses of 1, 5 or 10 mg/mouse. The rutin was dissolved in DMSO. The control groups of mice were injected with 0.2 ml 0.9% NaCl and 0.2 ml DMSO. Increasing doses of rutin 1, 5 or 10 mg per mouse caused augmentation of the tumor mass to 2400 mg, 2600 mg and 2800 mg respectively, whereas the tumor weight of the control group was 980 mg.
The median number of lung metastases in the control groups was 12; after treatment with 5 or 10 mg of rutin, the number of lung colonies increased to 19 and 27, respectively.
The administration of 10 mg rutin inhibited melanin formation by about 43%. The melanosomes in the experimental groups were in the 2nd or 3rd stage, and the low content of melanin was noticed.

Key words: Melanoma, rutin, metastasis, flavonoids, ultrastructure melanin content.
pp. 266-271