Electronic Library of Scientific Literature
Volume 43 / No. 6 / 1996
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
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
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
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
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
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
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
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
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
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
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