Electronic Library of Scientific Literature


Volume 47 / No. 2 / 2000



Myeloid differentiation and maturation of SCF+IL-3+IL-11 expanded AC133+/CD34+ cells selected from high-risk breast cancer patients

S. Filip, J. Vávrová, D. Vokurková, M. Bláha, J. Vaňásek

Department of Oncology and Radiotherapy, Charles University Hospital, 500 05 Hradec Kralové, Czech Republic, e-mail: filip@fnhk.cz;
Institute of Radiobiology and Immunology, Purkyně Military Medical Academy, Hradec Králové, Czech Republic;
Institute of Clinical Immunology and Allergology, Charles University Hospital, 500 05 Hradec Kralové, Czech Republic;
Department of Hematology, Charles University Hospital, Hradec Králové, Czech Republic

The AC133 antigen is selectively expressed on subset of CD 34+ cells isolated from leukapheresis products from high risk breast cancer patients receiving chemotherapy plus G-CSF. MiniMACS AC133+ isolated cells contained a mean of 85% (80-90) AC133+ cells. Enriched AC133+ cells coexpressed 80% CD34+, 6.6% CD33+ and 2% CD15+. Separated AC133+ cells contained 600 GFU-GM/104 cells and 70 BFU-E/104 cells. Flow-cytometric analysis indicated that AC133+ cells were isolated from cells population with low granularity (SS), while CD33+ a CD15+ cells had a high granularity. After a seven-day ex vivo expansion in the presence of SCF + IL-3 + IL11, the expansion of cells increased 19.4 times. The mean percentage of blasts decreased from 100% at the start of culture to 81% on day 3 and 30% on day 7. Promyelocytes were slow to appear with 10% present on day 3, but thereafter increased to 33% on day 7. The appearance of myelocytes and metamyelocytes lagged 3 days behind promyelocytes and continued to increase during culture to become the predominant (30%) cell type on day 7. Very few neutrophils (2%) were observed in any of the cultures on day 7. Monocytes or macrophages were not detected on day 7. By day 7 megakaryocytes were present at low levels (10%). The mean value of CFU-GM in the culture after day 7 of ex vivo expansion in the presence of SCF+IL-3+IL-11 had increased 45-fold, BFU-E 5-fold. After 7 days of expansion with IL-3+SCF+IL-11 cells expressed a mean of 12% CD34+, 8% AC133+, 59% CD33+ and 30% CD15+. The aim of this experiment was to determine whether ex vivo culture of peripheral blood AC133+ cells could generate sufficient numbers of progenitors to potentially abrogate cytopenia after transplantation and passive purging of tumor cells.

Key words: AC133+ cells, CD34+ cells, ex vivo expansion, myeloid lineage, intensive chemotherapy, breast cancer.
NEOPLASMA, 47, 2, 2000, 73-80


Differential modulation of xenobiotic metabolizing enzymes by vanadium during diethylnitrosamine-induced hepatocarcinogenesis in Sprague-Dawley rats

A. Chakraborty, S. Selvaraj

Bio-Sciences Division, Inter University Consortium for DAE Facilities, Calcutta-700 0091, India, e-mail: ani@alpha.iuc.res.in

Effect of vanadium on hepatic xenobiotic biotransformation in rats exposed to diethylnitrosamine (DENA, 200 mg/kg body weight, intraperitoneally) was investigated to elucidate a possible mechanism of vanadium mediated prevention of chemical carcinogenesis. Vanadium supplementation (0.5 ppm ad libitum with drinking water), at different phases before and after DENA treatment, significantly modulated the decrease in contents of total cytochrome P-450, cytochrome b5, activity of nicotinamide adenine dinucleotide phosphate (NADPH), (reduced form) cytochrome reductase, and uridine diphospho-glucuronyl transferase (UDPGT) in microsomal fractions of whole liver, hyperplastic nodules (HNs) and non nodular surrounding parenchyma (NNSP) as induced by DENA, 20 weeks following its administration. Supplementary vanadium had also substantial influence on the activities of cytosolic enzymes, like, uridine diphospho (UDP)-glucose dehydrogenase and NAD(P)H: quinone oxidoreductase (DT-diaphorase) in the concerned tissue which were observed to be remarkably decreased as a result of DENA treatment in comparison to that of the control counterparts. However, vanadium was found to have little or no effect on the lowering of aryl hydrocarbon hydroxylase (AHH) activity by DENA administration. On the basis of significant modulation of DENA induced alterations in cytosolic and microsomal enzyme activity it can be presumed that the chemoprotective effect of vanadium might be mediated through elevation of phase II conjugating enzymes which in turn, lead to a move and shift of metabolic profile that reduces the intracellular concentration of carcinogen derived reactive intermediates.

Key words: Diethylnitrosamine, hepatocarcinogenesis, rat liver, vanadium, xenobiotic biotransforming enzymes.
NEOPLASMA, 47, 2, 2000, 81-89


Characterization of factors inducing apoptosis in thymocytes of mice bearing a transplantable T-cell lymphoma of spontaneous origin

A. Shanker, S.M. Singh

School of Biotechnology, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh, India, e-mail: sukhmahendrasingh@yahoo.com

It has been observed that the progressive ascitic growth of a transplantable T-cell lymphoma of spontaneous origin in murine host, designated as Dalton’s lymphoma (DL), induces inhibition of various immune responses and is associated with an involution of thymus accompanied by a massive depletion of the cortical region and alteration in the distribution of thymocytes caused by induction of apoptosis with a decrease of CD4+CD8+, CD4+CD8- and CD4-CD8+ thymocytes. Here, we report that serum of DL-bearing mice contains soluble factors capable of inducing thymocyte apoptosis, the effectiveness of which increases with the progression of tumor growth. A decline of essential cytokines and hormones in the body due to their depletion by DL cells, which being a T-cell phenotype may have similar growth factor requirements, is ruled out by our results, suggesting additional apoptosis-inducing factors to be present in the tumor serum. Partial characterization of the serum to identify the biochemical nature of the putative serum-borne apoptosis inducing factor(s) showed that the same was proteinaceous. Further analysis of the sera of normal and DL-bearing mice by gel filtration using fast protein liquid chromatography (FPLC) and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that protein profile in the two sera differed quantitatively as well as qualitatively. FPLC analysis could resolve six peaks in both the sera, out of which the peak containing protein(s) in the range of MW 35 kD showed a higher magnitude and apoptotic activity followed by peaks containing proteins of MW in the range of 67 and 116 kD respectively as compared to that of the corresponding peaks in the normal serum. These observations were also confirmed by SDS-PAGE, with the resolution of additional proteins in the range of 25-26 kD which were found to be absent in normal serum. Further, the paper discusses different possible factors that could be associated with the progression of DL growth.

Key words: Dalton’s lymphoma, thymocytes, apoptosis-inducing factor(s).
NEOPLASMA, 47, 2, 2000, 90-95


Heterogeneity of dipeptidyl peptidase IV from C6 rat glioma cells

R. Malík, K. Vlašicová, L. Kadlecová, A. Šedo

Laboratory of Cancer Cell Biology, 1st Department of Medical Chemistry and Biochemistry, 1st Faculty of Medicine, Charles University, 121 08 Prague 2, Czech Republic; e-mail: Aleksi@mbox.cesnet.cz;
Laboratory of Cell Pathology, Institute of Experimental Medicine, Academy of Sciences, Prague, Czech Republic

Dipeptidyl peptidase IV is known to be involved, due to both hydrolytic and non-hydrolytic mechanisms, in various cell functions of normal and cancer cells as well. In this report dipeptidyl peptidase IV substrate and pH preferences, some inhibition parameters, freezing/thawing sensitivity and stability against hydrolysis by trypsin were studied in C6 rat glioma cells. Our results confirmed substantial heterogeneity of dipeptidyl peptidase IV population. Such observation is important to avoid methodological artifacts and to decrease risk of misinterpretations in biological studies.

Key words: Dipeptidyl peptidase IV, glial cells, enzyme inhibitors.
NEOPLASMA, 47, 2, 2000, 96-99


Cytotoxic activity of several unrelated drugs on L1210 mouse leukemic cell sublines with P-glycoprotein (PGP) mediated multidrug resistance (MDR) phenotype. A QSAR study

A. Breier, Z. Drobná, P. Dočolomanský, M. Barančík

Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, 833 34 Bratislava, Slovak Republic, usrdtylo@savba.sk;
Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovak Republic

L1210/VCR-1 and L1210/VCR-2 cell lines are multidrug resistant (MDR) sublines obtained by adaptation of mouse leukemic cell line L1210 to vincristine and, the development of MDR in these cell lines has been found to be associated with an overexpression of P-glycoprotein (PGP). In the present work we studied the relationship between the structure of 15 cytotoxic active substances (drugs) and their cytotoxicities on L1210/VCR-1 and L1210/VCR-2 resistant cell lines. The resistance of these MDR cells to the respective drugs was expressed as the ratio of IC50 values obtained for resistant and sensitive cells. These values of resistance were correlated with the following physico-chemical constants of the test substances: binding energy, Ebind; total energy of the molecule, Esum; aromaticity, Kpi; molecular weight, Mw; acidobasic constant, pKa; partition coefficient in water/octanol two phase system, log(p). It has been found that according to the cytotoxic effects the tested drugs may be divided into three groups: (i) drugs with higher cytotoxicity to the resistant cell lines as to sensitive cells (collateral hypersensitivity); (ii) drugs exhibiting approximately similar effects on sensitive and resistant cell lines; (iii) drugs with weaker cytotoxicity to resistant cells than to sensitive cells. No direct correlations with any physico-chemical constant described above could be established for cell resistance to the drug studied. However, resistance values could be fitted by multiple exponential regression with all described physico-chemical constants implied as six independent variables. The latter procedure made us to conclude that the ability of a drug to be a substrate for PGP is connected with its fulfilling the following criteria: (i) flexible structure of its molecule; (ii) molecular weight lower than ~1300 g/mol; (iii) nonprotonized character at pH 7.0.

Key words: P-glycoprotein, multidrug resistance, L1210 leukemic cell lines, cytostatic drugs, QSAR.
NEOPLASMA, 47, 2, 2000, 100-106


Content of epidermal growth factor receptor in metastatic breast cancer: its role in endocrine sensitivity prediction

Z.B. Nešković-Konstantinović, D.B. Nikolić-Vukosavljević, M.V. Branković-Magić, L.B. Mitrović, I.Spužić

Department Medical Oncology, Institute for Oncology and Radiology of Serbia, 11 000 Belgrade, Yugoslavia, e-mail: konsta@eunet.yu;
Laboratory for Receptors and Biology of Malignant Cells, Institute for Oncology and Radiology of Serbia, Belgrade, Yugoslavia;
Department for Research and Education, Institute for Oncology and Radiology of Serbia, Belgrade, Yugoslavia;
Serbian Academy of Sciences, 11 000 Belgrade, Yugoslavia

Epidermal growth factor receptor (EGF-R) is known as an indicator of endocrine independence of breast cancer. However, a small proportion of EGF-R expressing tumors was found to respond to endocrine treatments. On the other side, a cut-off point of EGF-R positivity is not yet defined. In the aim to find out whether there exists a cut-off value that sharply discriminate the endocrine sensitive and endocrine insensitive breast cancers, the quantitative EGF-R content was analyzed in a group of 42 female patients with metastatic disease, being routinely treated with chemo-, chemo-endocrine, or endocrine therapy alone. Steroid receptors (SR) and EGF-R were determined by biochemical methods in tissue samples of an unselected group of patients. Patients with metastatic disease, either at diagnosis, or developed after the treatment of operable or locally advanced breast cancer, were included in the present analysis. According to the treatments used, and their therapeutic response, all patients were divided in endocrine sensitive or resistant, and chemo-sensitive or resistant. The SR and EGF-R status and content was analyzed in relation to the sensitivity to both systemic treatments. The EGF-R content was significantly lower in responders to endocrine treatments, compared to non-responders, while there was no difference in EGF-R level, in relation to the sensitivity to chemotherapy. In addition, the EGF-R content was significantly higher in chemo-sensitive tumors, than in endocrine sensitive. On the contrary, ER content was significantly higher in endocrine sensitive, than in endocrine resistant, and in chemo sensitive patients, as well. Similar differences were found in PR content, but they were less pronounced. While the individual ER contents in endocrine sensitive and endocrine resistant tumors overlapped, the EGF-R ranges were different: no one endocrine sensitive tumor exceeded the EGF-R content of 26 fmol/mg, thus suggesting the EGF-R cut-off point of endocrine sensitivity. The clinical use of EGF-R, with the cut-off point of 26 fmol/mg, in addition to clinical criteria of endocrine sensitivity and SRs, would significantly improve the correct endocrine sensitivity prediction (from 52 to 78%). In conclusion, in a group of metastatic breast cancer patients, treated routinely by systemic therapies it was found, that the use of higher cut-off point for EGF-R positivity can improve the prediction of endocrine sensitivity. The prognostic relevance of this cut-off value remains to be analyzed.

Key words: Breast cancer, endocrine treatment, steroid receptors, EGF-R, predictive factors.
NEOPLASMA, 47, 2, 2000, 107-113


A fraction isolated from Ehrlich ascites carcinoma as an antitumor and differentiating agent against human leukemic cell ML-2

P. Sur, N. Nandi, P. Ghosh, N.C. Ghosh

Department of Pharmacology and Experimental Therapeutics, Indian Institute of Chemical Biology, Jadavpur University, Calcutta - 700032, India, e-mail: iichbio@sias101.vsnl.net.in;
Department of Physics, Jadavpur University, Calcutta, India;
Department of Chemistry, Jadavpur University, Calcutta, India

Lipopolysaccharide fraction isolated from Ehrlich ascites carcinoma (E-LPS) was investigated as an antitumor agent against human leukemia cell ML-2. Marked cell growth inhibition was observed with ML-2 cell accompanied by inhibition of DNA synthesis and perturbation of cell cycle. Induction of differentiation in treated ML-2 cells was observed as indicated by morphological maturation, NBT reducing activity and indirect immunofluorescence.

Key words: Ehrlich ascites carcinoma, LPS fraction, ML-2 cell, differentiation.
NEOPLASMA, 47, 2, 2000, 114-117


Electrophoretic characterization of a gamma-1-heavy chain disease

M. Tichý, J. Stulík, J. Ošanec, P. Škopek

Purkyně Military Medical Academy, 500 01 Hradec Králové, Czech Republic, e-mail: tichy@fnhk.cz;
Department of Clinical Biochemistry, Military Hospital, Plzeň, Czech Republic;
Department of Hematology and Oncology, Charles University, Plzeň, Czech Republic

This report describes a new case of gamma-1-heavy chain disease found in a woman with malignant lymphoproliferative disease. The patient’s serum and urine containing gamma-1-heavy chains were analyzed using different electrophoretic approaches, especially two-dimensional electrophoresis and immunoblotting analysis. In a serum sample, five sets of gamma-1-heavy chain spots differing in molecular weight with acidic pI values and one set of more basic gamma-1-heavy chain spots were found. The major group of spots exhibited molecular weight in the range from 29 to 39 kDa. Examination of urine sample proved the presence of the more basic set of gamma-1-heavy chain spots and two acidic groups, including 29 to 39 kDa set.

Key words: Heavy chain disease, two-dimensional polyacrylamide gel, electrophoresis, immunoblotting, monoclonal gamma heavy chains.
NEOPLASMA, 47, 2, 2000, 118-121


Paraquat-induced lipid peroxidation and injury in Ehrlich ascites tumor cells

P. Karakashev, L. Petrov, A. Alexandrova

Institute of Physiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria, e-mail: phristov@bio.bas.bg

The participation of lipid peroxidation products in the mechanisms of paraquat toxicity in Ehrlich ascites tumor (EAT) cells was observed. Paraquat in a concentration 0.5-1.0 mmol increased the level of lipid peroxidation according to the Ohakawa TBARS (thiobarbituric acid-reactive substances) method. These changes in TBARS production in EAT cells correlated with paraquat toxicity on the cells registered by using the method for cell injury, which is based on changes in lactate dehydrogenase activity. The metal chelator DTA removed the effect of paraquat on TBARS production and on cell injury. The present data suggested that the increased level of lipid peroxidation and cell injury is a result of the paraquat action in EAT cells depending on iron.

Key words: Lipid peroxidation, cells injury, tumor, paraquat, iron.
NEOPLASMA, 47, 2, 2000, 122-124


Hematological and cytogenetic response of interferon alpha 2b alone and combined interferon alpha plus cytarabine as a first-line treatment in chronic myeloid leukemia

E. Tóthová, M. Fričová, A. Kafková, N. Štecová, T. Guman, Š. Raffač, M. Hlebašková

Department of Hematology, Medical Faculty Hospital and UPJŠ, 040 66 Košice, Slovak Republic, e-mail: etothova@post.sk

Treatment with interferon-alpha (IFNalpha) prolongs survival in chronic myeloid leukemia (CML). Additionally, cytarabine (AraC) can reduce the number of Ph + metaphases.
Fortythree previously untreated patients with CML in chronic phase were randomly assigned to receive either. IFNalpha 2b (5 MU sqm/daily) or IFNalpha 2b in the same dosages plus monthly courses of low-dose AraC. The aim were complete hematologic remission at 6 months and cytogenetic response at 12 months.
A complete hematologic remission occurred in 60.4% patients with single IFNalpha 2b in 76.2% patients with combination therapy. A cytogenetic response was present in 13.9% (major in 2 patients) with IFN therapy and in 38.1% patients with combination therapy. Two of 21 patients treated with IFNalpha/AraC therapy achieved major (9.52%), 4 partial (19.04%) and 2 minor (9.52%) cytogenetic response. Major side effects were cytopenia (20.1%), flu-like syndromes (42.4%) and increase of hepatic transaminases (3.4%). The side effects were more significant in the group receiving combination therapy.
Based on published data that show a survival advantage for patients who achieved any cytogenetic response, and high rate of cytogenetic response which we observed in our study we believe that IFN plus AraC regimen could be a front-line therapy for CML.

Key words: Chronic myeloid leukemia, interferon, cytarabin, hematologic response, cytogenetic response.
NEOPLASMA, 47, 2, 2000, 125-128


Radiotherapy of early stages Hodgkin’s disease. 10 years experience of the Masaryk Memorial Cancer Institute

J. Petera, H. Machařová, R. Pohanková, A. Malíř, P. Čoupek, M. Konečný, J. Patera, J. Pecina, J. Drbal, H. Koukalová, I. Vášová

Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic, e-mail: petera@fuhk.cz.;
Faculty Hospital Bohunice, Brno - Bohunice, Czech Republic

Radiotherapy and chemotherapy, alone or in combination, are curative treatment methods in early stages of Hodgkin’s disease (HD). The choice of treatment depends on the stage of the disease, histological type and localization of the tumor, as well as on other prognostic factors. A retrospective study was conducted including 145 patients with clinical Stages I and II of HD according to Ann Arbor classification, all treated in the Masaryk Memorial Cancer Institute in Brno during the years 1985 through 1994. 80 patients were males (55%) and 65 patients females (45%). The age of the patients ranged from 11 to 77 years, with an average of 34.8 years. 41 patients were diagnosed with Stage IA tumor, 1 patient with Stage IB, 75 patients with Stage IIA and 28 with Stage IIB disease. The histological types of the disease were lymphocyte predominant in 23 patients, nodular sclerosis in 49 patients, mixed cellularity in 65 cases and lymphocyte depletion in 8 cases.
91 patients were treated with radiotherapy alone. In this group 14 patients relapsed within the radiation field (15%) and 25 outside the radiation field (28%). 39 patients were treated with combination of radiotherapy and chemotherapy. In this group relapse occurred within the radiation field in 3 patients (8%) and outside the radiation field in 7 patients (18%). 15 patients were given chemotherapy alone, 7 patients from this group experienced a relapse.
The five-year survival was 81% in patients with Stages IA and IIA disease, 65% in Stages IB and IIB disease. The five-year survival in the patients who relapsed was 56%.
Radiotherapy remains the curative method of choice in highly selected group of patients with early stages of Hodgkin’s disease.
The results of radiotherapy alone are unsatisfactory in unselected clinical Stage I-II patients because of the presence of patients with adverse prognostic factors, particularly B symptomatology, mixed cellularity/lymphocyte depletion histology, higher age. These patients are candidates for combined treatment. Modern equipment and meticulous treatment are conditions crucial for the outcome of curative radiotherapy in patients with Hodgkin’s disease.
Combination chemotherapy is very effective in the treatment of relapse following the primary radiotherapy.

Key words: Hodgkin’s disease, early stages, radiotherapy, chemotherapy, relapse, survival.
NEOPLASMA, 47, 2, 2000, 129-132


The impact of delivered dose errors on local control of irradiated advanced laryngeal cancer

L. Miszczyk, R. Tarnawski, K. Składowski

Radiotherapy Department, Institute of Oncology, M. Skłodowska-Curie Cancer Center, 44-100 Gliwice, Poland, e-mail: leszek@onkol.instonko.gliwice.pl

On the basis of 1015 entrance and 863 exit dose in vivo measurements, 863 calculations of midline dose were done, and the average deviation and ranges of its value were estimated. Data of 710 advanced larynx cancers were reviewed in order to achieve dose-response relationship. Patients data were fitted directly to L-Q model using maximum likelihood estimation. In 16.5% of measurements the deviation of midline dose was larger than -5.2%. A steep dose-response relationship for TCP was found. Considering -5.2% deviation of 2 Gy fraction and 72 Gy of total dose, the 17% (from 48 to 31%) decrease of TCP was found. It shows that deviations of delivered dose influence the tumor control probability and that after systematic error finding during fractionated radiotherapy the value of remaining fraction size and total dose should be modified to compensate the change of TCP.

Key words: Dosimetry in vivo, dose errors, tumor control probability, laryngeal cancer.
NEOPLASMA, 47, 2, 2000, 133-136