Electronic Library of Scientific Literature


Volume 43 / No. 6 / 1996

Placental isoferritin-associated p43 in pregnancy and breast cancer

Harald R. Rosen

Ludwig Boltzmann Research Institute for Surgical Oncology, Donauspital/SMZ-Ost, A-1220 Vienna, Austria

Ferritin, physiologically an iron-storage protein, has been repeatedly investigated with regard to its role in the development of inflammation and malignancy [2, 3, 6]. The structural heterogeneity of this protein has aroused considerable interest in recent years [2, 3, 5, 6]. Although ferritin is generally regarded as an iron-storage protein, small amounts are also found in the sera of normal individuals, while abnormally high concentrations are found in the serum of patients with malignancies as well as in pregnant women.
In the past, considerable attention has also been paid to the structural heterogeneity of ferritin derived from various organs and malignant tissues [3, 5]. It has been reported that the placenta, fetal tissues and malignant tissues contain acidic ferritin, while the liver and the spleen contain ferritin in the basic form. The acidic isoform has been summarized under the term oncofetal ferritin, since these forms share certain physical-chemical characteristics.
These so-called oncofetal or placental ferritins (PLF) have repeatedly been shown to have an inhibitory effect on hematopoiesis and T-cell function [2, 6, 7, 10].
The purpose of the present review is to elucidate current data concerning the role of placental isoferritin in pregnancy as well as in the development of breast cancer.

pp. 357-362

Immunosuppressive activity of lymphocyte mitogenesis by breast cancer-associated p43

M. Reinerova, Z. Veselovska, C. Ausch, H.R. Rosen

Cancer Research Institute of the Slovak Academy of Sciences, 812 32 Bratislava, Slovakia;
Ludwig Bolzmann Research Institute for Surgical Oncology, University of Vienna, Vienna, Austria

Placental isoferritin (PLF), an acidic isoform of ferritin, and its unique superheavy chain of 43 kDa (p43) has been described to be synthesized by human breast cancer cells. Physiologically, p43 PLF produced by the placenta is involved in immune suppression of maternal lymphocytes aimed at fetal antigens. A study was carried out to elucidate a paradigm of p43 occurrence in breast cancer patients. Immunosuppression of cytotoxic CD8+ lymphocytes was measured via inhibition of blast transformation in concanavalin A (ConA) stimulated peripheral blood lymphocytes (PBL) using [^3]H-thymidine uptake in vitro. PBLs were cultivated from 29 women having benign lesions in the breast as well as from 41 patients with breast adenocarcinoma. In breast cancer patients addition of p43 significantly inhibited the activation of lymphocytes proliferation by ConA compared to women with benign tumors. The addition of indomethacin or levamisole did not influence this inhibitory effect of p43 in breast cancer patients. Presence of interleukin-2 in cultures was able to overcome the inhibitory effect of p43 on CD8+ lymphocytes proliferation from women having breast adenocarcinomas and to increase its value in patients with benign lesions.

Key words: Breast cancer, lymphocyte cultures, concanavalin A, immunosuppression, p43.
pp. 363-366

Leukemia-associated phenotypes: Their characteristics and incidence in acute leukemia

O. Babusikova, M. Glasova, E. Konikova, J. Kusenda

Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia

Leukemia-associated phenotypes have been suggested to be a valuable tool for the detection of minimal residual disease in acute leukemia patients, as they allow to distinguish leukemic blasts from normal hematopoietic progenitor cells. The aim of the present study was to analyze the proportion of acute leukemia patients (both with lymphoid and myeloid leukemias) in which the immunological detection of leukemia-associated phenotypes was convenient for the distinction of leukemic and normal cells. For this purpose we have studied the blast cells from 186 acute leukemia patients at diagnosis with a large panel of monoclonal antibodies by flow cytometry using double staining combinations. From aberrant phenotypes on blast cells we followed lineage infidelity (coexpression of myeloid markers in lymphoid leukemia cells and vice versa, as well as the simultaneous expression of both, T and B cell markers in one lymphoid blast cell) and asynchronous marker expression (simultaneous expression of early and late markers in one cell). One hundred and five of the 186 acute leukemia cases analyzed (56%) showed the presence of leukemia-associated phenotypes. In 41 of the 90 ALL cases followed (46%) and in 40 of the 96 AML cases studied (42%) lineage infidelity was observed. Asynchronous antigen expression was detected in 24 followed cases (13%).
Evaluation of the cell marker density by means of calibration microbeads demonstrated abnormal mean channel immunofluorescence and molecules of equivalent soluble fluorescein for CD8 in two patients with T cell malignancies at diagnosis. Abnormal CD8 density might thus represent a characteristic feature of malignant CD8-positive T cell clone. Quantitative marker evaluation therefore seems to be another important mean for the detection of aberrant phenotypes on leukemia cells suitable for the detection of minimal residual disease.

Key words: Leukemia-associated phenotypes, minimal residual disease, acute leukemia, quantitative flow cytometry.
pp. 367-372

Some early differentiation markers detected in cytoplasm of pre-B cells by flow cytometry

E. Konikova, M. Glasova, J. Kusenda, O. Babusikova

Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia

Immunofluorescent staining of cytoplasmic IgM (heavy chains) and CD24 as well as their simultaneous staining with surface B cell markers was used to study immunophenotype changes in B cell differentiation. Human hematopoietic B cell lines P3HR1 and RAJI were used. We found that IgM and CD24 cell markers while absent on cell membrane could be detected in their cytoplasm (c). The presence of cIgM in cell lines RAJI, P3HR1 indicates their early pre-B differentiation stage. The presence of cCD24 simultaneously with mCD22 and cIgM is the evidence that hematopoietic cell lines or leukemias may not accurately reflect normal differentiation pathway.
Combinations of cIgM, cCD24 with surface B cell markers CD10, CD19 on these cell lines can be considered as leukemia associated phenotypes. Some of them were shown in bone marrow and peripheral blood of pre-B ALL and B-CLL patients and can be used for the detection of minimal residual disease.
Different fixation/permeabilization methods were tested in order to choose the optimal one for simple detection of cytoplasmic markers or their simultaneous detection with surface markers by flow cytometry. They included "one-component-methods" (methanol - M, saponin - S), methods combining these components with paraformaldehyde (P+M, P+S) or buffered formaldehyde acetone (BFA). The choice depended on individual marker detected. General parameters like the proportion of debris, cell aggregation, cell loss and the changes of scatter parameters FSC and SSC were taken into consideration. The priorities of combined methods P+S, P+M1 and BFA over one-component methods are demonstrated.

Key words: Flow cytometry, simultaneous immunofluorescent staining, cytoplasmic marker, cell differentiation, human hematopoietic cell lines, cell fixation and permeabilization.
pp. 373-380

Flow cytometric detection of some activation and proliferation markers in human hematopoietic cell lines

M. Glasova, E. Konikova, J. Kusenda, O. Babusikova

Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia

Simultaneous surface marker/DNA, cytoplasmic/DNA or nuclear/DNA staining was used to study proliferation of hematopoietic cell lines (MOLT4, BJAB, P3HR1). Different fixation/permeabilization methods (paraformaldehyde with metanol or Tween 20 or saponin, buffered formaldehyde-acetone) were used providing optimal results of the double stainings. There was a significant increase of S phase and proliferation index (PI) of CD71+ and Ki67+ MOLT4 cells in comparison with their negative counterparts. This indicates their close connection with proliferation. Unlike that, the correlation between the expression of CD38 and S phase or PI was not significant either in MOLT4 or in P3HR1 cells.
For cytoplasmic markers CD3 (in MOLT4 cells) and CD22 (in BJAB cells) statistically significant (cCD3) and not significant (cCD22) correlation was demonstrated between their expression and S phase or PI.
Molecular equivalents of soluble fluorescein values for CD71 were always higher than for CD38. The density of these cell surface markers in addition to the percentage of their expression is of considerable significance for their evaluation as activation or proliferation markers.

Key words: Double staining, cell activation and proliferation, fixation/permeabilization method, cytoplasmic marker, Ki-67, S phase, antigen density.
pp. 381-388

Cell surface phenotype and increased penetration of human multidrug-resistant ovarian carcinoma cells into in vitro collagen-fibroblasts matrix

J. Sedlak, O. Sedlakova, P. Hlavcak, L. Hunakova, J. Bizik, M. Grofova, B. Chorvath

Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia;
National Cancer Institute, Bratislava, Slovakia

Human multidrug resistant ovarian carcinoma cells (A2780/ADR) exhibited increased in vitro penetration into the collagen - normal human fibroblasts matrix, increased cell surface expression of alpha[_6] integrin (CD49f antigen) and slightly increased expression of alpha[_2] (CD49b) integrin compared with that of parental drug-sensitive A2780 cells. Both, multidrug-resistant and parental, drug-sensitive, cell lines did not express the 67 kDa non-integrin high affinity laminin receptor on their cell surfaces. As there were no marked differences between metalloproteinase activity of both A2780 cell sublines (with similar intensity of 72 kDa and 92 kDa lysis bands in zymograms), the increased penetration of the drug-resistant subline into the collagen-fibroblast gel matrix might be associated with the increased expression of adhesion proteins (including collagen-binding alpha[_2] integrin), or cell surface-associated collagenase-stimulating protein(s). This multidrug resistant ovarian carcinoma cell line might serve as an in vitro model of neoplastic cells with increased biological aggressiveness, molecular mechanisms of which require further analysis.

Key words: Ovarian carcinoma cell line - multidrug-resistance - adhesion antigens - CD49b - alpha[_2]-, CD49f - alpha[_6] integrins - 67 kDa laminin receptor - metalloproteinases MMP-2, MMP-9 - collagen-fibroblasts matrix - E-cadherin - biological aggressiveness.
pp. 389-395

The study of AgNOR proteins in leukemias: Diagnostic value and correlation to S-phase cell fraction

M. Klobusicka, O. Babusíkova, A. Mesarosova, J. Kusenda, M. Glasova

Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia

The study assessed the diagnostic value of silver staining method and its possible relevance as an alternative to DNA analysis for the study of cellular proliferation in various leukemias (ALL, AML, CML). Silver staining of nucleolar organizer region-related proteins (AgNORs) was applied to peripheral blood and bone marrow cells. The analysis of S-phase cells was carried out using a FACStar flow cytometer. The mean number of AgNOR dots per nucleus and the percentage of S-phase cells varied according to immunophenotype of leukemic cells, depending on the time of initial diagnosis, remission or relapse. Peripheral blood and bone marrow cells of healthy subjects exhibited less AgNOR dots than leukemic cells. The number of AgNORs in bone marrow cells was higher than that of AgNORs in peripheral blood. Significant differences between ALL and AML, as well as AML and CML in AgNOR quantity were observed. Important increase in AgNOR values was evident in relapsed leukemias and in the CML blast crisis. DNA flow cytometry analyses provided results comparable to those of AgNOR enumeration. The correlation between AgNOR dots and proportion of S-phase cells prompted us to consider that AgNOR count reflects cell proliferation capacity of leukemic cells.

Key words: Leukemia, AgNOR proteins, S-phase cells, diagnosis.
pp. 397-401

Potential carcinogenity of the synthetic 1,3,6-triazine (6-aza) nucleic acid analogues determined by DC polarography I. Nucleobases

L. Novotny, A. Vachalkova, A. Piskala, R. Petrasova, B. Blesova

Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia;
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic;
Institute of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic

The polarographic reduction of several synthetic 1,3,6-triazine (6-aza) nucleobases in the strictly anhydrous solution was studied in the absence and presence of alpha-lipoic acid. The values of the half-wave potentials E[_1/2] and the parameter of potential carcinogenicity tg alpha were determined for one natural and 5 synthetic nucleobases. The current value of the first diffuse polarographic wave or a new diffuse polarographic wave belonging to the nucleobase-alpha-lipoic acid complex increased with the increased alpha-lipoic acid concentration for the all compounds only marginally. Although this diffuse current increase was linearly depended on the alpha-lipoic acid concentration in anhydrous solutions, the determined index tg alpha values ranging between 0.029 and 0.108 indicated a very low potential carcinogenicity of the all nucleobases investigated.

Key words: 1,3,6-triazine nucleobases, 6-aza nucleobases, thymine, reduction potentials, carcinogenicity, DC polarography, parameter tg alpha.
pp. 403-406

Comparison of the in vitro effect of adriblastina on induction of SCEs in V79 cells and human peripheral blood lymphocytes

E. Szabova

Institute of Preventive and Clinical Medicine, Department of Genetics, 833 01 Bratislava, Slovakia

Induction of sister chromatid exchanges (SCEs) by the cytotoxic antibiotic adriblastina (doxorubicin, 14-hydroxyrubidomycin) of the anthracycline group isolated from Streptomyces peucetius var. caesius by Farmitalia Research Laboratories was tested in vitro at concentrations of 0.01 microg/ml, 0.1 microg/ml and 0.2 microg/ml using V79 cells and human peripheral blood lymphocytes from healthy donors. In comparison with negative control, adriblastina significantly elevated the SCE frequency both in V79 cells and in human peripheral blood lymphocytes. The results obtained by comparing the effect of equivalent adriblastina concentrations on V79 cells and human peripheral blood lymphocytes showed no significant difference in the mutagenicity effect of both of these cell lines to adriblastina.

Key words: Adriblastina, V79 cells, human lymphocytes, SCE induction.
pp. 407-409

Approaches to prove the melanoma origin in amelanotic human melanoma cell lines

M. Blasko, I. Chalupa, J. Borovansky, M. Tocekova, J. Siracky

Department of Cytogenetics and Tumor Biology, Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia;
2nd Institute of Chemistry and Biochemistry, 1st School of Medicine, Charles University, Prague, Czech Republic;
School of Science' Department of Genetics and Molecular Biology, Comenius University, Bratislava, Slovakia

The human melanoma B-HM8 cell line was derived from highly pigmented malignant skin melanoma. After 5 weeks of cultivation it entirely lost the pigmentation and has remained amelanotic since. Electron microscopy revealed neither premelanosomes nor melanosomes and the cells did not release detectable amount of dopa-oxidase activity into culture medium. Immunocytochemical studies using the polyclonal anti-S-100 antibody and detection of alpha-mannosidase activity in culture medium proved the melanoma origin of B-HM8 cells. Chromosomal changes in the karyotype of these cells were typical for human melanoma with chromosomes No. 1, 5, 7, 9, and 11 involved most frequently.

Key words: Human melanoma B-HM8 cell line, immunocytochemistry, cytogenetics, tyrosinase and alpha-mannosidase activity.
pp. 411-415

Protective effects of cardioxane against anthracycline-induced cardiotoxicity in relapsed acute myeloid leukemias

P. Lemez, J. Maresova

Institute of Hematology and Blood Transfusion, 128 20 Prague 2, Czech Republic;
1st Department of Internal Medicine, 1st Medical Faculty, Charles University, Prague, Czech Republic

The clinical use of anthracyclines and related antitumor agents is limited by their cumulative dose-related cardiac toxicity. Cardioxane (ICRF-187) is an agent that has been recommended to block selectively this toxicity which e.g. limits the use of daunorubicin (DNR) in doses higher than 550-700 mg/m[^2]. We decided to use cardioxane in patients with relapsed acute myeloid leukemias (AML) who have previously been treated with DNR doses above 500 mg/m[^2]. Seven patients with relapsed AML received cardioxane 30 min before DNR or mitozantrone (MTZ) in doses 8-13 x higher than DNR or 40-60 x higher than MTZ. Two patients received anthracyclines cumulative doses corresponding to more than 1300 mg/m[^2] and 1000 mg/m[^2] of DNR, respectively, without any signs of cardiac toxicity. The other 5 AML patients in relapse received 1-3 chemotherapy cycles with cardioxane. Their total cumulative doses of DNR were 550-750 mg/m[^2] and their left ventricular ejection fraction remained above 50% as were their pretreatment values. Cardioxane seems to be a useful cardioprotective agent in relapsed AML which enables further treatment with anthracyclines.

Key words: Acute myeloid leukemia, cardioxane, ICRF-187, cardioprotection, anthracycline-induced cardiotoxicity.
pp. 417-419