Electronic Library of Scientific Literature


Volume 47 / No. 6 / 2000


Reliability and limitations of cytochemistry in diagnosis of acute myeloid leukemia - Minireview

M. Klobušická

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovak Republic, e-mail: exonklo@savba.sk

Accurate characterization of leukemic blast cells is an important prerequisite of the precise diagnosis of acute myeloid leukemia and has a great impact on therapy and prognosis. The purpose of this review is to consider the present possibilities and limitations of enzyme cytochemistry and to emphasize how cytochemistry may contribute to the final classification and differential diagnosis of acute myeloid leukemia. The role of conventional enzyme cytochemistry, either dominant or subsidiary, in the discrimination of acute myeloid leukemia subgroups is discussed. The survey confirms the necessity of immunological marker analysis in the accurate diagnosis of minimally differentiated myeloid leukemias and acute leukemia of megakaryocytic lineage. In these cases, the cytochemical evaluation provides insufficiently relevant information regarding blast cell origin. On the other hand, cytochemical investigation is appreciated to be dominant over immunophenotyping in characterizing majority of acute myeloid leukemia subgroups, because of the availability of standardized and sufficiently specific cytochemical reactions and, because of the lack of specificity of the many of immunological markers against myeloid antigens. The immunocytochemical, cytogenetic, molecular biology and electron microscopic studies shortly mentioned in this review supplement the information for correct diagnosis of acute myeloid leukemia.

Key words: Acute myeloid leukemia, enzyme cytochemistry, immunofenotyping.

Neoplasma, 47, 6, 2000, 329-334


A double germline mutations in the APC and p53 genes

V. Zajac, M. Tomka, D. Ilenčíková, P. Májek, V. Števurková, T. Kirchhoff

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovak Republic, e-mail: exonvzaj@savba.sk;
National Cancer Institute, Bratislava, Slovak Republic

Germline mutation in the APC gene is required for the initiation of the development of familial adenomatous polyposis (FAP). According to Fearon and Vogelstein model, further somatic mutations in the K-ras oncogene, DCC gene and p53 tumor suppressor gene are prerequisite for development of colon carcinoma. We have found that the germline mutations in the DNA isolated from lymphocytes of an 18 years old girl with extraordinary expressive phenotype in codons 1060-1061 of the APC gene result in truncation of the APC protein. The mutation in codons 12 and 13 of the K-ras oncogene was not detected, but another germline mutation was found in codon 210 of the p53 gene. Furthermore, no one of these germline mutations was detected in the DNA of peripheral blood lymphocytes of the patient’s 21 years old healthy sister. Until now, there has been no evidence about the expressive phenotype due to mutation in codons 1060-1061 of the APC gene; the role of germline missense mutation in codon 210 of the p53 gene in the FAP malignant process remains to be elucidated too. The effect of the combination of germline mutation in two different tumor suppressor genes in the progress of disease is discussed.

Key words: Familial adenomatous polyposis (FAP), APC gene, p53 gene, mutation detection, heteroduplex analysis (HDA), protein truncation test (PTT).

Neoplasma, 47, 6, 2000, 335-341


Expression of the non-classical HLA-G antigen in tumor cell lines is extremely restricted

K. Poláková, G. Russ

Cancer Research Institute, Slovak Academy of Sciences, 833 91, Bratislava, Slovak Republic, e-mail: exompola@savba.sk;
Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic

It has been proposed that tumor cells frequently associated with partial or total loss of HLA class Ia expression may abnormally express HLA-G class Ib antigen. Such peculiar HLA class I expression would allow tumor cells to escape not only from CD8+T but also from NK-cell cytotoxicity. We studied the cell surface expression of HLA-G using flow cytometry with two HLA-G specific monoclonal antibodies (87G, 01G). The JEG-3 choriocarcinoma cell line, which constitutively expresses HLA-G antigens was used as a positive control. We did not detect the cell-surface HLA-G antigens in the following 75 tumor cell lines: melanoma (22), neuroblastoma (7), retinoblastoma (1), glioma (2), breast carcinoma (3), ovarian carcinoma (3), cervical carcinoma (1), colon carcinoma (3), bladder carcinoma (2), hepatocarcinoma (1), sarcoma (2) and leukemia cell lines: T-lymphocytes (6), B-lymphocytes (13) and myelo-monocytes (9). We found that some myelo-monocytic cell lines express on their surface high affinity FcgammaRI (CD64) that may result in the binding of HLA-G specific mabs to their cell surface even in the absence of HLA-G molecules. Our panel of HLA-G negative tumor cell lines accommodated 62 cell lines for which similar analysis have not been reported and also contained 13 cell lines with total or partial loss of HLA class Ia molecules. Our observation imply that under normal culture conditions the cell surface HLA-G reactive with 87G and 01G mabs is absent in most tumor cell lines of different origin.

Key words: HLA class I, HLA-G, tumor cell lines, FcgammaRI.

Neoplasma, 47, 6, 2000, 342-348


Oxidative/antioxidative effects of different lignin preparations on DNA in hamster V79 cells

D. Slameňová, B. Košíková, J. Lábaj, Ľ. Ružeková

Cancer Research Institute,Slovak Academy of Sciences, 833 91 Bratislava, Slovak Republic, e-mail: exonslam@savba.sk;
Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic

Endogenous oxidative damage to DNA is thought to be an important etiologic factor in the development of chronic diseases such as cancer. Many products of the vegetable kingdom have been suggested to limit oxidative damage to DNA in humans. To this group belong lignins, polyphenols present in all plants (including edible plants). The aim of this study was to examine oxidative/antioxidative effects of different lignin preparations on mammalian DNA. In addition to a water-soluble sulfur-free lignin 1 which was obtained by fractionation of hardwood hydrolysate, we investigated lignin 2 (obtained by oxidation of lignin 1), lignin 3 (prepared by the extraction of lignin 2 with a mixture ethanol-water 3:1), lignin 4 (Na-salt of lignin 3) and lignin 5 (prepared by extraction of lignin 2 with diethylether). Our results showed that only the original lignin 1 did not increase substantially the level of DNA damage. Lignins 2, 3, 4 and 5 increased both the level of frank DNA strand breaks + alkali-labile sites and the level of FPG-sensitive sites representing oxidative damage to DNA. Lignin 1 was further tested for its antioxidative activity against DNA base modifications generated by visible light+photosensitizer. Obtained results confirmed the oxygen species-scavenging activity of lignin 1.

Key words: Hamster cells V79, oxidative damage to DNA, FPG-sensitive sites, single cell gel electrophoresis, five different preparations of lignin.

Neoplasma, 47, 6, 2000, 349-353


Failure of carboxymethylglucan to inhibit oxidative DNA damage induced by hydroxyl radicals or singlet oxygen

A. Gábelová, M. Plešková

Cancer Research Institute of Slovak Academy of Sciences, 833 91 Bratislava, Slovak Republic, e-mail: exongaba@savba.sk

The ability of carboxymethylglucan (CMG), a high molecular water-soluble derivative of glucan, was evaluated to act as a scavenger of reactive oxygen species. Hydrogen peroxide and methylene blue plus visible light, well-defined oxidant factors, were used as a model agents for induction of oxidative DNA damage in CaCo-2 cells. Both hydrogen peroxide and visible light gave rise to dose-dependent increase of DNA damage mediated by hydroxyl radicals (OH) or singlet oxygen (1O2), respectively. While DNA lesions generated by hydrogen peroxide dominated by strand breakage, exposure of CaCo-2 cells to visible light led mainly to base modifications sensitive to formamidopyrimidine DNA-glycosylase (Fpg). Neither CMG nor ascorbic acid, a known antioxidant, induced any DNA damage in CaCo-2 cells. Pretreatment of cells with ascorbic acid prior to H2O2 or visible light exposure resulted into statistically significant reduction of DNA lesions induced by particular agent. However, pretreatment of CaCo-2 cells with CMG in concentration range from 0.01 microM to 1 microM reduced neither the level of strand breaks induced by hydrogen peroxide nor the number of Fpg-sensitive base modifications generated by visible light.

Key words: Carboxymethylglucan, hydroxyl radical, singlet oxygen, ascorbic acid, single cell gel electrophoresis.

Neoplasma, 47, 6, 2000, 354-361


Co-expression of GFAP, vimentin and cytokeratins in GL-15 glioblastoma cell line

A. Peržeľová, I. Máčiková, M. Tardy, P. Mráz, J. Šteňo, I. Bízik

Department of Anatomy, School of Medicine, Comenius University, 813 72, Bratislava, Slovak Republic, e-mail: Perzelova@fmed.uniba.sk;
INSERM U-421, IM3, Creteil, France;
Department of Neurosurgery, Dérer Hospital, Bratislava, Slovak Republic

Glial fibrillary acidic protein (GFAP), vimentin (Vi) and cytokeratin (CK) intermediate filament (IF) proteins were studied in glioblastoma cell line GL-15. The immunofluorescence staining revealed strong positive staining for vimentin in all cultured cells. Approximately 20% of analyzed cells showed strong and 50% moderate intensity of staining for GFAP. About 3% of all cells were positively stained with a mixture of anti-CK monoclonal antibodies. The expression of all IF was not in relation to the cell density or days in vitro after passage. The double immunofluoresce revealed that all CK-positive cells express GFAP and vimentin. This study demonstrates the heterogeneity of the clonal GL-15 glioma cell line which consists in three immunocytochemically distinct cell types: Vi+/GFAP-/CK-, Vi+/GFAP+/CK-, and Vi+/GFAP+/CK+. These findings give further evidence about the expression of non-glial IF in cultured glioma cells.

Key words: Intermediate filaments, glioma cell line, keratin, vimentin, GFAP.

Neoplasma, 47, 6, 2000, 362-366


Radiation-induced DNA damage and repair evaluated with ‘Comet assay’ in human ovarian carcinoma cell lines with different radiosensitivities

G. Bačová, Ľ. Hunáková, M. Chorváth, E. Bolješíková, B. Chorváth, J. Sedlák, A. Gábelová

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovak Republic, e-mail: exonbaco@savba.sk;
Department of Radiotherapy, St Elisabeh Cancer Institute, Bratislava, Slovak Republic

Radiation-induced DNA damage and kinetics of DNA repair was evaluated in three human ovarian carcinoma cell lines (i.e. CH-1, A-2780 and SKOV-3) with different sensitivities to ionizing radiation and radiation-induced apoptosis with the aid of single cell gel electrophoresis (SCGE, the comet assay). A good correlation was found between the initial level of DNA breaks and radiation induced apoptosis in CH-1 and SKOV-3 cell lines. While the radiation-sensitive CH-1 cell line manifested the highest level of initial DNA breakage and a significant delay in DNA break rejoining, the inverse correlation was found in the radiation-resistant cell line SKOV-3. Intermediate initial level of breaks was induced in the A-2780 cell line characterized by the intermediate sensitivity to X-ray radiation in comparison to CH-1 and SKOV-3 cells, however, the kinetics of DNA repair was comparable with radiation-resistant cell line SKOV-3. Our data suggest that the comet assay could be a promising tool for prediction of intrinsic cell radiosensitivity. This method might be considered as a supplementary technique to the more reliable but time consuming clonogenic assay.

Key words: Human ovarian carcinoma cell lines, single cell gel electrophoresis (the comet assay), flow cytometry, radiation-induced DNA damage, DNA repair kinetics, radiosensitivity.

Neoplasma, 47, 6, 2000, 367-374


Mismatch repair in Xenopus egg extracts is not strand-directed by DNA methylation

M. Petranović, K. Vlahović, D. Zahradka, S. Džidić, M. Radman

Department of Molecular Genetics, Ruđer Bošković Institute, 10000 Zagreb, Croatia, e-mail: dina@rudjer.irb.hr;
Faculté de Médecine Necker-Enfants Malades, Université R. Descartes, Paris Cedex 15, France

The efficiency of Xenopus laevis egg extract to repair T:G and A:C mismatched base pairs in unmethylated, hemimethylated and fully methylated heteroduplexes was investigated. Filamentous phage M13mp18 and its derivative M13mp18/MP-1 (C changed to T inside the sequence dCC*C GGG, at the position 6248) were used for heteroduplexes construction. The three origins of mismatched base-pairs in the eukaryotic DNA are mimicked by in vitro methylation: hemimethylated DNA (me-/me+) for replication errors; unmethylated (me-/me-) and fullymethylated DNA (me+/me+) for recombination heteroduplexes, and fullymethylated also for locally, spontaneously deaminated 5-methylcytosine (5meC) to T, generating the exclusively T:G mismatch. The methylations were in CpG dinucleotides, mostly characteristic of eukaryotic cells [5, 24].
In vitro methylation was done by HpaII methylase which methylate central C of dCCGG sequence in the manner of eukaryotic methylation. The position of mismatched bases was chosen so that correction of mismatched bases in any strand would create the sequence for one of the ”diagnostic” restriction endonucleases, either BstNI or MspI.
Correction efficiency was about 108 repair events per egg equivalent. Correction in favor of C:G base pair restoration occurred regardless of the T:G or C:A mispairs, with almost equal efficiency. Repair of T:G to T:A was up to 10 times less efficient comparing to C:G, and repair of C:A to T:A was in our experimental system undetectable. No significant difference in repair efficiency of mismatched bases situated in unmethylated, hemimethylated or fullymethylated heteroduplexes indicate methylation-independent repair of mismatched bases in X. laevis oocite extracts.

Key words: Mismatch repair, heteroduplex DNA, DNA methylation, strand discrimination.

Neoplasma, 47, 6, 2000, 375-381


Correlation of clinical picture (event free survival and overall survival) in childhood acute leukemia patients with immunophenotype and chromosomal abnormalities

O. Babušíková, D. Sejnová, G. Kirschnerová, Z. Kiršnerová, J. Čáp

Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovak Republic, e-mail: exonobab@savba.sk;
Department of Childhood Oncology, University Children´s Hospital, Bratislava, Slovak Republic;
National Cancer Institute, Bratislava, Slovak Republic

In a group of 102 children with different immunological subtypes of acute leukemia, both lymphoblastic and nonlymphoblastic, the clinical parameters – event free survival and overall survival were correlated with numerical and structural chromosomal abnormalities. In a group of 80 ALL patients genetic abnormalities were observed in 40 patients, from those 19 of numerical type, 17 of structural type and 4 with both, numerical and structural anomalies. From the whole ALL group observed 23 patients (28.75%) died. In 10 died patients genetic abnormalities were found and in 6 cases less mature T-phenotype ALL has been documented. It seems, therefore, that immature T-phenotype with pathological karyotypes of all types of genetic anomalies presents the most risk group of patients of which all children died. ALL patients, as a whole, with pathological karyotype have shown significantly lower event free survival rate, comparing to the group of ALL patients with normal karyotype. Overall survival rate was also lower in the first group, but statistically not significant. In T-ALL patients, in both groups, with and without pathological karyotype, event free survival rate and overall survival rate were also lower in the first group, but statistically not significant. In B-ALL patients with pathological karyotypes vs. normal ones overall survival rate was lower in the first group, but statistically not significant. There was no difference in overall survival rate in these patients between pathological and normal karyotypes. In ANNL group of patients pathological karyotype was observed in 14 of them, with numerical anomalies in 6 patients, structural in 4 patients and both of them – numerical + structural in 4 children. From the whole ANLL group observed 11 (50%) patients died during the follow-up period (9 in relapse and 2 of treatment complications). From 11 died patients in 81.8% pathological karyotype was present. The prevalence of pathological karyotypes was observed in less mature M0-M2 ANLL subtype (71.4%). ANLL patients with pathological karyotype have shown significantly lower event free survival rate (in one of the two statistical log-rank analyses), comparing to the group of ANLL patients with normal karyotype. Overall survival rate was also lower in the first group, but statistically not significant. The presence/absence of CD34 marker expression in blast cells of our group of acute leukemia patients did not show any difference in event free survival and overall survival rates.

Key words: Acute leukemia in children, overall survival and event free survival, immunophenotype, chromosomal abnormalities.

Neoplasma, 47, 6, 2000, 382-389


Diazenes as modificators of drug-resistance in tumor cells

M. Osmak, T. Bordukalo, A. Ambriović Ristov, B. Jernej, J. Košmrlj, S. Polanc

Department of Molecular Genetics, Ruđer Bošković Institute, HR-10 000 Zagreb, Croatia, e-mail: osmak@rudjer.irb.hr;
Faculty of Chemistry and Chemical Technology, SI-1000 Ljubljana, Slovenia

To overcome the drug resistance, which is the major obstacle in the successful treatment of cancer patients, various compounds have been tested. Glutathione is one of the most promising targets for modulation. In the present study, we examined the influence of five new synthesized compounds – diazenes on the reduction of the intracellular level of GSH. Further, we investigated their ability to increase the cytotoxicity of cisplatin, vincristine and doxorubicin. In experiments human parental cervical (HeLa) and laryngeal (HEp2) carcinoma cells and their drug-resistant cell sublines (HeLaCA and CK2, respectively) were used. Intracellular GSH content was examined spectrophotometrically by the procedure developed by Tietze. The cell sensitivity to drugs was determined using a modified colorimetric MTT assay. Results showed that the rate of reduction of GSH concentration was dependent on the cell type and the type of diazenes. We did not find a correlation between the reduction in GSH level and increased cytotoxicity to selected anticancer drugs. Nevertheless, we found that: a) diazenes LV-35 and VZ-19 increased the cytotoxicity of cisplatin in HEp2 cells, b) diazene MG-19 potentiated the cytotoxicity of vincristine in HEp2 cells, and c) diazene VZ-19 in HeLaCA cells. These data suggest that specific combination of diazene and anticancer drug may be useful in the treatment of certain tumor types.

Key words: Tumor cells, drug resistance, diazenes, glutathione.

Neoplasma, 47, 6, 2000, 390-395


Quality of life in cancer patients treated by chemotherapy

M. Scieszka, M. Zielinski, M. Machalski, Z.S. Herman

Clinic of Internal Diseases and Oncological Chemotherapy Silesian Medical Academy, 40-029 Katowice, Poland, e-mail: pskm@polbox.com;
Department of Clinical Pharmacology Silesian Medical Academy, Katowice, Poland

Many studies connected with different aspects of the quality of life (QL) have been increasingly reported. In this study we have been used FACT questionnaire to estimate QL in three groups of cancer patients receiving chemotherapy. Questionnaires were completed by 177 patients. Adverse effects of treatment severely influence the cancer patients QL. The most significant worsening of QL was noticed in the group of lung cancer patients receiving the most emetogenic chemotherapy(i.e. cis-platinum, vepesid). In other two groups (gastric and colorectal cancer patients) side effects of chemotherapy caused relatively less QL deterioration. This finding implicates possibility of intensifying the course of treatment (dosage and duration) assuming there is no hematopoetic insufficiency.

Key words: Quality of life, cancer patients, gastric cancer, colorectal cancer, lung cancer.

Neoplasma, 47, 6, 2000, 396-399


CD44 and its v6 spliced variant in lung carcinomas: Relation to NCAM, CEA, EMA and UP1 and prognostic significance

Viet Nhung Nguyen, T. Miřejovský, L. Melínová, V. Mandys

Hlava Institute of Pathology, First Faculty of Medicine, Charles University, Prague, Czech Republic;
Pneumological Clinic, First Faculty of Medicine, Charles University, Prague, Czech Republic;
Department of Teratology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 128 00 Prague, Czech Republic, e-mail: vmand@lf1.cuni.cz

CD44 is a polymorphic family of cell surface glycoproteins that was recently reported to have important role in cell adhesion and migration as well as modulation of cell–matrix interactions. Thus, expression of CD44 has been proposed to be associated with malignant behavior of tumors like invasive growth and formation of metastasis. The expression of CD44s and its v6 isoform (CD44v6) was determined immunohistochemically in 106 lung tumors of various histophenotypes, degrees of differentiation, and clinical stages. The results were compared with the expression of NCAM, CEA, EMA and UP1 and with clinicopathological parameters including patients’ survival. CD44s was expressed in all histophenotypes of non-small cell lung carcinomas (NSCLC) with tendency being squamous cell lung carcinoma (SqCC) > bronchioloalveolar adenocarcinoma (BAC) > conventional adenocarcinoma (ConAC) (91, 66.7 and 38.9%, respectively). Almost identical distribution of positivity revealed CD44v6 in all three subgroups of NSCLC mentioned above (91, 66.7 and 36.1%, respectively). In the subgroup of neuroendocrine tumors, CD44s and CD44v6 were restrictedly expressed in small cell lung carcinomas (2/14 tumors), while all 3 typical carcinoids were strongly positive for these markers. Expression of NCAM and CEA was significantly higher in adenocarcinoma subgroup than those in SqCC subgroup (45.7 and 75% vs. 14.8 and 39%, respectively). NCAM expression was also significantly different in BACs and in ConACs (69.2 vs. 36.4%, p < 0.05). The expression of CD44 was related to the differentiation of SqCC. The carcinomas with keratinization were CD44 positive. Adenocarcinomas producing mucin were CD44 negative. The expression of CD44, NCAM, CEA, EMA and UP1 did not correlate with lymph node metastasis and disease stage. CD44V6 was the only marker that its expression was closely related to patients’ survival. The absence CD44v6 but not CD44s in NSCLC group was associated with significantly longer survival of patients compared to patients with CD44v6 positive tumors. This difference was even higher in tumors negative for CD44v6 and simultaneously NCAM and/or CEA positive. The data of this study suggest that CD44v6 might be an independent prognostic factor in NSCLC. Moreover, our data give another evidence of diverse role of CD44 in the differentiation and progression of non-small cell lung carcinomas and neuroendocrine carcinomas of the lung.

Key words: CD44, NCAM, CEA, EMA, UP1, immunohistochemistry, lung cancer.

Neoplasma, 47, 6, 2000, 400-408


Colorectal carcinoid tumors – own experience

M. Bartos, J.M. Narębski, K. Kaczka, L. Pomorski

Clinic of Endocrinological and General Surgery, Institute of Endocrinology, Medical University of Łódź, 93-513 Łódź, Poland, e-mail: branch@poczta.onet.pl

Symptomatology, diagnostics and treatment problems in 5 patients with colorectal carcinoid are presented. From 1974 to 1999 in the Clinic of Endocrinological and General Surgery of the Medical University of Łódź, 3001 patients underwent surgery due to acute appendicitis and 431 for colorectal cancer. Among them, there were 5 patients in whom the histological examination revealed colorectal carcinoid. The carcinoids were localized in the appendix in 4 patients and in the left colon flexure in 1 patient. The mean age of these 5 carcinoid patients at the time of diagnosis was 38.4 years (range 18-72 yr). The female-to-male ratio amounted to 4:1.
The symptoms of all 5 patients was not typical for carcinoid of the colon. In four surgery was performed for acute appendicitis and one patient complained of chronic obstipation and pain in the left epi- and mesogastrium. The double-contrast examination of the large intestine revealed tumor of the left colon flexure.
Four carcinoid patients with the signs of acute appendicitis had emergency surgery. The carcinoid tumors were diagnosed microscopically after surgery only. In 3 of them the tumor extended beyond the appendix and a reoperation was performed. In one patient with the tumor of small diameter (5 mm) involving only the mucosa and submucosa a reoperation was not indicated. In 3 reoperated patients right hemicolectomy with regional lymphadenectomy was performed. The patient with the tumor of the left colon flexure diagnosed preoperatively underwent radical surgery with regional lymphadenectomy. The postoperative histological examination of the tumor confirmed carcinoid. No carcinoid metastases were found in lymph nodes of all studied cases.
Until today, all 5 carcinoid patients are alive with no signs of local reccurrence or distant metastases over the 1-20 year follow-up period.

Key words: Colorectal carcinoid, apudoma.

Neoplasma, 47, 6, 2000, 408-412