Electronic Library of Scientific Literature


Volume 45 / No. 5 / 1998

Hematopoietic cell differentiation antigens (CD system 1997). Cancer research relevance - Minireview

B. Chorváth, J. Sedlák

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

The 6th International Workshop on Leukocyte Antigens (white cell differentiation antigens) continued the international cooperative effort aimed at characterization of all leukocyte cell surface antigens with respect to their biochemical properties, cell- and cell line expression, molecular and cellular function(s) and eventual disease relevance.
Significantly, among 36 newly defined CD clusters identified with the aid of numerous monoclonal antibodies submitted to the workshop [17], 8 new clusters belonged to the endothelial section, 6 new CD clusters were identified within cytokine receptor section and 5 such clusters were characterized in the adhesion structure section, i.e. as antigens with the pattern of expression also on cells, tissues and cell lines outside the hematopoietic system (Tables 1-3). Minority of newly defined clusters appurtened to lineage-specific or non-lineage hematopoietic differentiation antigens (Table 4), i.e. 5 new CD clusters within myeloid section, 3 new clusters within both non-lineage and NK antigens, 2 T-cell antigens and one new CD cluster defined within both B-cell or platelet sections.

Key words: Hematopoietic cell, cancer research, antigens, CD system 1997.
pp. 273-276

To the incidence of nucleoli in circulating myeloblasts of patients suffering from acute myeloblastic, promyelocytic and myelomonocytic leukemias

K. Smetana, H. Šubrtová, I. Jirásková, H. Klamová, P. Lemež, L. Rosa

Institute of Hematology and Blood Transfusion, 128 20 Prague 2, Czech Republic;
Medical Faculty Hospital Královské Vinohrady, Prague, Czech Republic

Nucleoli were studied in circulating myeloblasts of myeloblastic (FAB M1, M2), promyelocytic (FAB M3) and myelomonocytic (FAB M4) acute myeloid leukemias (AMLs) using a cytochemical procedure for the demonstration of RNA. In patients untreated with cytostatic chemotherapy, myeloblasts of myeloblastic acute leukemias possessed less frequently "active large" nucleoli and more frequently "inactive" micronucleoli in comparison with other investigated types of AMLs. When myeloblasts were classified according to the presence of functionally dominant nucleoli, the higher percentage of "terminal" myeloblasts containing only micronucleoli in this type of AML was significantly reduced in patients treated with the cytostatic chemotherapy. In patients suffering from promyelocytic leukemia treated with cytostatic chemotherapy, the decreased percentage of myeloblasts containing functionally dominant active large nucleoli was accompanied by the increased incidence of myeloblasts with functionally dominant "resting" ring shaped nucleoli. In myelomonocytic AML no significant differences were noted between patients untreated or treated with the cytostatic chemotherapy in the incidence of main nucleolar types in myeloblasts and myeloblasts classified according to the presence of functionally dominant nucleoli. Thus a further biological specificity might exist among leukemic blasts in various types of AMLs which should be considered for a rational approach to the therapy of these malignancies. In addition, the cytostatic chemotherapy did not influence incidence of the nucleolar asynchrony in myeloblasts of all investigated types of AML.

Key words: Nucleoli in circulating leukemic myeloblasts.
pp. 277-281

Intracellular markers in acute myeloid leukemia diagnosis

E. Koníková, M. Glasová, J. Kusenda, O. Babušíková,

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

In our study we used a new proposed system of CD45 monoclonal antibody in combination with the side scatter (SSC) parameter as a very useful gating method allowing myeloblast detection especially in cases with low blasts percentage in examined samples.
Immunological demonstration of myeloperoxidase (MPO) in the cytoplasm of AML blasts is considered to be a reliable and highly sensitive marker. Using a direct single and double immunofluorescence staining method and flow cytometry we evaluated the intracellular expression of two granular constituents of myeloid cells - MPO and lactoferrin (LF) in leukemia cells from 18 patients at AML diagnosis, two patients in remission after allogenic bone marrow transplantation and in six controls. Two different fixation/permeabilization techniques were used: Fix&Perm, paraformaldehyde and saponin prior to monoclonal antibody staining in order to verify the sensitivity of two labeling methods for MPO. Although both reagents used in this study proved to be efficient tools for the fixation and permeabilization of leukemia cells, the second one was characterized by higher sensitivity in detection of MPO. By double staining of MPO and LF we were able to distinguish undifferentiated cells from the granulomonocytic maturation compartments in bone marrow, since LF is proposed to be selectively expressed from the myelocyte stage of differentiation onward. Cytoplasmic CD13 expression was detectable in AML blasts after their buffered-formaldehyde-acetone fixation/permeabilization. According to our results the detection of MPO and CD13 markers in the cytoplasm of leukemia cells is of great importance in the definition of FAB M0-M1 subtype of AML.
Furthermore we described overexpression of CD34 antigen in AML and revealed the characteristic marker combination when CD34 was studied simultaneously with MPO. This finding also coincided with some atypical phenotypic features (CD15/MPO, CD7/cCD13, CD2/cCD13, CD33/cCD13, MPO/cCD13) contributing to the differential diagnosis and allowing the immunologic monitoring of patients for the presence of residual disease.

Key words: Acute myeloid leukemia, CD45-SSC gating, immunophenotyping, fixation/permeabilization, myeloperoxidase, lactoferrin, cytoplasmic CD13, quantitative flow cytometry.
pp. 282-291

Cytogenetic study of acute myeloid leukemia: Comparison of data obtained in 1991-1996 and 1982-1988

J. Musilová, K. Michalová, Z. Zemanová, Z.Březinová, A. Dohnalová, J. Sajdová

3rd Medical Department, 1st Faculty of Medicine and Faculty Hospital, 128 08 Prague 2, Czech Republic;
Institute of Hematology and Blood Transfusion, Prague, Czech Republic;
The Biostatistical Institute of the 1st Medical Faculty, Prague, Czech Republic

The results of the cytogenetic study of bone marrow cells from 110 consecutive patients with primary acute myeloid leukemia (AML) who were diagnosed and treated between 1991 and 1996 at one tertiary care institution were compared with similar data obtained between 1982 and 1988 in 130 patients. Despite improvements in cytogenetic techniques (namely FISH methods, applied in all patients with abnormal karyotypes since 1990) in recent years we have observed a significantly lower frequency of abnormal karyotypes: 52.7% versus 77.7% (p = 0.001). This was mainly due to the decreased frequency of patients with +8, -5, -7 and inv(16). The survival rate (excluding the patients who underwent a bone marrow transplantation) was only slightly increased.

Key words: Acute myeloid leukemia, chromosomes, survival, FISH.
pp. 292-295

Pentoxifylline stimulates drug-induced apoptosis in leukemic cells

P. Rauko, J. Sedlák, J. Duraj, M.F. Szekeres, L. Novotný

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia;
Clinical Institute for Medical and Chemical Laboratory Diagnostics, University of Vienna, Vienna, Austria;
Faculty of Pharmacy, Kuwait University, Safat, 13110 Kuwait

Camptothecin (CAM) and cisplatin (cis-diamminedichloroplatinum(II), cis-Pt) were used as inducers of apoptosis in the mouse leukemic L1210 cells. Relatively high concentrations of 50 µmol cis-Pt and 50 µmol CAM, respectively, were used to induce the apoptotic DNA ladder. The simultaneous treatment of L1210 cells by the drug and pentoxifylline (PTX) resulted in a decrease of drug concentrations necessary for the induction of apoptosis. This study revealed that a cell cycle G2 checkpoint inhibitor PTX reduces time intervals necessary for the onset of drug-induced apoptosis in these cells. This fact might be important as the earlier onset of programmed cell death may decrease a risk of tumor cells to become resistant to drug therapy.

Key words: Pentoxifylline, camptothecin, cisplatin, cell proliferation, flow cytometry, apoptosis.
pp. 296-300

Proliferating cell nuclear antigen (PCNA) expression in gestational trophoblastic diseases (GTD)

J. Molykutty, T.N. Rajalekshmy, N.M. Balaraman, E. Swapna, N.M. Krishnan, P. Balaram

Division of Cancer Research, Regional Cancer Centre, Trivandrum - 695 011, Kerala, India;
SAT Hospital, Medical College, Trivandrum, India;
Doctors Diagnostic and Research Centre, Ulloor, Trivandrum, India

Gestational Trophoblastic Disease is an abnormal condition of the placenta, the incidence of which is very high in the state of Kerala, India. The proliferative rate of molar placentas in comparison with the normal placentas of comparable gestational age group was done in order to find out its role in the prognosis of this tumor by assessing the expression of PCNA in trophoblasts. PCNA expression was evaluated in 149 trophoblastic tumors and 96 normal placental tissue. The percentage of positive cells was significantly increased in molar placentas of the 1st trimester in comparison to the normal placentas. Correlation of the staining score to the regression pattern of the tumor showed a significant increase in the chemotherapy group when compared to the spontaneously regressing group. But no correlation was found between the percentage of PCNA positive cells with histological grade of the tumor proliferation.

Key words: Proliferating cell nuclear antigen, GTD, placenta.
pp. 301-304

Combined therapy of B16(F10) murine melanoma using E.coli cytosine deaminase gene and murine interleukin-4 gene

E. Missol-Kolka, A. Sochanik, S. Szala

Department of Tumor Biology, Institute of Oncology, 44-100 Gliwice, Poland

This paper summarizes preliminary results of combining suicide gene strategy (E.coli cytosine deaminase gene - CD) with immunotherapy (murine interleukin-4 gene) for treatment of experimental B16(F10) melanomas implanted into C57Bl/6 mice. The best therapeutic results, inhibition of tumor growth and prolonged survival time of treated vs. control mice, were obtained when plasmid expression vectors containing therapeutic genes were transferred into mice via DDAB/DOPE cationic liposome carrier on the third or fourth day following inoculation of mice with cancer cells. Extension of survival time has been noted in the case of two-gene therapy (as compared with one-gene therapy) of tumors which originated from cells transfected in vitro with CD gene and which were subsequently injected in vivo with IL-4-secreting cells. However, no improvement of therapeutic effect was obtained in case of mice treated with a combination of two genes transferred intratumorally with DDAB/DOPE cationic liposomes as compared to mice treated with a single gene only.

Key words: Gene therapy, cationic liposomes, suicide genes, interleukin-4, E.coli cytosine deaminase, combined therapy.
pp. 305-311

Genetic polymorphism of glutathione S-transferases M1 and T1 as a risk factor in lung and bladder cancers

J. Šalagovič, I. Kalina, J. Štubňa, V. Habalová, M. Hrivňák, L. Valanský, A. Kohút, E. Biroš

Department of Medical Biology, School of Medicine, P. J. Šafárik University, 040 66 Košice, Slovakia;
Department of Tuberculosis and Respiratory Diseases, Teaching Hospital, Košice, Slovakia;
Department of Urology, Teaching Hospital, Košice, Slovakia;
Department of Pharmacology, School of Medicine, P. J. Šafárik University, Košice, Slovakia

A combined analysis of two polymorphic enzymes, glutathione S-transferase µ (GST M1) and q (GST T1) and their implication as cancer risk factors was performed in a case-control study of lung and bladder cancers. Using a multiplex polymerase chain reaction (PCR) based method, the frequency of the homozygous deleted GSTM1 and GSTT1 genotypes was examined in 117 lung cancer patients, 67 urinary bladder cancer patients, and in a community-based sample of 248 healthy, unrelated individuals. In both cancer groups the frequency of the GSTM1 null genotype was higher in comparison with that of the control group (59% and 59.7% vs. 49.6%), but this increase did not reach statistical significance (p > 0.05). After grouping by the smoking status, among smokers in both cancer groups (62.1% in lung cancer and 71.4% in the bladder cancer group, respectively) there were statistically significantly (p < 0.05) increased frequencies of the GSTM1 deletion genotype as compared to the control group (49.6%). Smokers with absence of the GSTM1 gene were at an approximately 1.7-fold higher risk for lung cancer (odds ratio - OR = 1.67, 95% confidence interval - CI 95% = 1.0-2.7, p = 0.04) and an approximately 2.5-fold higher risk for bladder cancer (OR = 2.54, CI 95% = 1.2-5.5, p = 0.02). As related to GSTT1, our study demonstrated an overall GSTT1 effect on bladder cancer risk. Individuals with absence of the GSTT1 gene were at an approximately 2.5-fold higher risk of developing bladder cancer. In the lung cancer cases, the frequency of the putatively high risk GSTT1 null genotype was not increased as compared with controls. No effect of smoking was found on risk of lung and bladder cancer associated with the GSTT1 0/0 genotype. In combined analysis, the obtained results suggested that individuals who were both GSTM1 null and GSTT1 null may be at increased risk because they lack both enzymes. The findings suggest that the GSTM1 null genotype may be associated with susceptibility to lung and urinary bladder cancer in dependence on the exposure to carcinogens in cigarette smoke and that the GSTT1 null genotype is not a critical factor in mediating the risk of lung cancer, but may be associated with an increased susceptibility to bladder cancer.

Key words: Genetic polymorphism, glutathione S-transferases M1 and T1, cancer risk, susceptibility, lung and bladder cancers.
pp. 312-317

Cysteine proteases and cysteine protease inhibitors in non-small cell lung cancer

E. Křepela, J. Procházka, B. Kárová, J. Čermák, H. Roubková

Department of Molecular and Cellular Pneumology, Clinic of Pneumology and Chest Surgery, Medical Faculty Hospital Bulovka, 180 71 Prague 8, Czech Republic;
Department of Chest Surgery, Clinic of Pneumology and Chest Surgery, Medical Faculty Hospital Bulovka, Prague, Czech Republic;
Department of Pathology, Medical Faculty Hospital Bulovka, Prague, Czech Republic

In this study we investigated the levels of two lysosomal cysteine protease proteins cathepsin B (CB) and cathepsin L (CL) and the levels of three cysteine protease inhibitor proteins stefin A (SFA), stefin B (SFB) and cystatin C (CNC) in squamous-cell lung carcinoma (SQCLC) and matched lung parenchyma specimens and examined the inhibition of CB and cathepsin C (CC) activities by endogenous inhibitors in extracts from SQCLC, lung adenocarcinoma (LAC) and lung parenchyma specimens. We found that Stage I SQCLCs contained significantly increased levels of CB protein, CB activity and SFA protein as compared to matched lungs. Neither the levels of CL protein nor the levels of SFB protein nor the levels of CNC protein in Stage I SQCLCs and the lungs were significantly different, but the levels of CB and CL proteins as well as the levels of SFA and SFB proteins showed significant positive correlation in SQCLCs. In SQCLCs as well as in the lungs the level of SFB protein was significantly higher than the level of SFA protein or the level of CNC protein. In the lungs the levels of SFA protein and CNC protein revealed a weak negative correlation trend. In extracts from SQCLCs the level of SFA protein showed a weak negative correlation with the residual CB activity (i.e. the activity remaining after extract preincubation) whereas in extracts from the lungs the level of CNC protein displayed a weak negative correlation trend with the residual CB activity and with the residual CC activity. We observed that SQCLCs and LACs contained not only a significantly increased activity of CB but also a significantly higher inhibitory potential against the activity of endogenous CB as compared to matched lungs. Leupeptin, a small inhibitor of CB, was capable to protect CB in lung carcinoma and lung parenchyma extracts from preincubation-induced inhibition, revealing an active-site directed and competitive nature of CB inhibition by endogenous cystatins. Ultrafiltration passaged protein preparations of nominal Mr =< 30 000 obtained from extracts of SQCLCs inhibited significantly higher quantities of activity of purified bovine spleen CC than did such protein preparations from matched lungs. Reaction courses of purified bovine spleen CC that had been preincubated with such protein preparations resembled those of endogenous CC from SQCLC and lung extracts showing a slow steady-state approach. These observations and the relaxation kinetics of CC from SQCLC and lung extracts suggest that CC in the extracts may be complexed with some cystatins. In conclusion, our results indicate that quantitatively different combinations of cystatins are the major constituents of the inhibitory potential against CB and CC in SQCLCs and the lungs.

Key words: Non-small cell lung carcinoma, cathepsin B, cathepsin C, cathepsin L, stefin A, stefin B, cystatin C, cysteine protease inhibitors.
pp. 318-331

Prognostic relevance of non-Hodgkin's lymphomas cell cycle data

J. Vučković, G. Forenpoher, M. Marušić, B. Užarević, T. Zemunik

Department of Hematology, Split Health Center, Split, Croatia;
Department of Pathology, Split University Hospital, Split, Croatia;
Department of Immunology, Zagreb University School of Medicine, Zagreb, Croatia;
Department of Clinical Laboratory Diagnosis, Zagreb University School of Medicine and University Hospital Centre, Zcgreb, Croatia;
Department of Biology, Split University School of Medicine, 21 000 Split, Croatia

Determination of proliferative activity of non-Hodgkin's lymphomas (NHL), aimed at improving the prediction of their clinical behavior, has gained considerable attention in the recent years. Flow cytometry has allowed rapid measurement of the cellular DNA content in terms of ploidy and proliferative activity.
Flow cytometric DNA analysis was performed on paraffin embedded biopsy specimens taken from 125 patients with NHL. In 90 of them, proliferative index (PI) could be accurately measured and correlated with histology grade of the Working Formulation (WF). Intermediate and high grade NHL (54 patients) were analyzed together as HG-NHL. With the discrimination point for PI of 10%, the survival of high and low proliferative lymphomas was compared in the whole NHL group and within the WF prognostic groups.
The median PI was 5% in LG (low grade) NHL and 10% in HG (high grade) NHL group. Acturial survival in NHL with high proliferative activity (39 patients) was 31% at 5 years and 15% at 10 years, and in NHL with low proliferative activity (51 patients) 53% and 18%, respectively (p = 0.002). In HG-NHL, survival at 5 years for low proliferative cases was 55% and for high proliferative cases 28% (p = 0.065), whereas in the LG-NHL group it was 54% and 28%, respectively (p = 0.059). The survival at 10 years was nearly equal in all groups.
Proliferative index was associated with the overall survival of NHL in the whole group, as well as within the LG and HG prognostic categories. PI could differentiate more and less aggressive NHLs both within LG-NHL and HG-NHL. A tendency of survival curves toward continuous relapse was observed in low proliferative NHL and a tendency toward "plateau" in high proliferative NHL, irrespective of the histology grade.

Key words: Non-Hodgkin's lymphomas, flow cytometry, proliferative index, survival.
pp. 332-335

Candida parapsilosis fungemia in cancer patients - incidence, risk factors and outcome

V. Krčméry Jr., S. Špánik, S. Grausová, J. Trupl, I. Krupová, A. Roidová, T. Šálek, J. Šufliarsky, J.Mardiak

Department of Medicine, School of Public Health, University of Trnava, Trnava, Slovakia;
Department of Pharmacology, St. Elisabeth Cancer Institute, 812 50 Bratislava, Slovakia;
National Cancer Institute, Bratislava, Slovakia;
Department of Chemotherapy, Department of Geriatrics, Postgradual Medical School, Bratislava, Slovakia

The paper presents an analysis of fungemia cases which were caused by C. parapsilosis in a cancer center within 10 years, with the aim to compare risk factors and the outcome with fungemias caused by C. albicans and other non-albicans Candida spp. fungemias.
Before 1990 (1988-1989) in our institutes C.parapsilosis fungemias were not observed at all. During 1990-1997, the proportion of C.parapsilosis among fungemias increased, in 1990-1993 from 0% to 7.1% in 1996-1997 to 14.2-15%. It represents 25% out of non-albicans Candida spp. fungemias and 7.9% out of all fungemias and is the third commonest pathogen after C. albicans (50.5%) and C.krusei (9.9%). Two from eight (25%) C.parapsilosis fungemias were breakthroughs, one appeared during prophylaxis with ketoconazol and one with fluconazol. Considering the proportion of C.parapsilosis among blood cultures, 13 of 170 blood cultures contained C. parapsilosis (6.6% among all yeasts from blood cultures). C.parapsilosis was the second commonest fungal organism isolated from blood cultures (after C. albicans) in our cancer center.
Infected vascular catheters were surprisingly not the major risk factor: central venous catheters were documented as a source in two cases only. The commonest risk factors were similar to those occurring with other fungemias - such as preceeding antimicrobial therapy (62.5%), neutropenia (50%) and prior prophylaxis with azoles.

Key words: Candida parapsilosis, cancer patients, fungemia, incidence, risk faktors.
pp. 336-344