Electronic Library of Scientific Literature


Volume 42 / No. 5 / 1995

Gene therapy for cancer (Present status). Minireview


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

The present status of cancer gene therapy is reviewed here in short. Two of the main gene therapy strategies for the treatment of cancer are discussed. The first main strategy is direct gene therapy which involves insertion of a functioning tumor suppressor gene or suppression of expression of a known oncogene. The second main strategy is indirect gene therapy which involves the insertion of a gene that modifies the cell to be more immunogenic for the host. The main clinical gene therapy trials are reviewed in their present state, including the replacement of defective tumor suppressor genes, the insertion of suicide or sensitivity genes, the insertion of prodrug-activating genes, and the use of virally directed enzyme prodrug therapies. Other topics discussed are the protection of stem cells from toxic effects of chemotherapy and new directions for gene therapy of neoplastic disease.

Key words: Cancer gene therapy, oncogene, tumor suppressor gene, retroviral vector, suicide gene, clinical trials.
pp. 209-213

Galectin-3, a laminin binding protein, fails to modulate adhesion of human melanoma cells to laminin


Metastasis Research Laboratory, University of Liege, B-4000 Liege, Belgium;
¹Molecular Pathology Section, Laboratory of Pathology, National Cancer Institute, Bethesda, USA;
²Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, USA

Galectin-3 is a laminin binding protein which expression is altered in a variety of human carcinomas including colon, breast and endometrium. In these tumors, we consistently observed a down regulation of galectin-3 expression related to increased aggressiveness. Galectin-3 belongs to a family of galactose-binding lectins and binds laminin through its numerous poly-N-acetyllactosamine chains. To date, the exact role of galectin-3 in the complex interactions between cancer cells and laminin has not been clearly defined. Adhesion of melanoma cells to laminin is a critical event during tumor invasion and metastasis. In this study, we explore the possibility that galectin-3 could modulate attachment of two human melanoma cell lines to laminin. A2058 and A375 melanoma cell expressed galectin-3 on their surface as demonstrated by immunofluorescence, and attached to laminin in an in vitro assay. We demonstrate that neither recombinant galectin-3 nor an affinity purified antigalectin-3 antiserum altered adhesion of A2058 or A375 melanoma cells to laminin. Our data strongly suggest that galectin-3 is not a key element in adhesion of the melanoma cells to laminin. These results are not surprising in light of the observation that galectin-3 expression is down regulated in cancer and that increased adhesion to laminin is a constant feature of invasive cancer cells.

Key words: Laminin, adhesion, galectin-3, invasion, metastasis, melanoma.
pp. 215-219

Immune phenotype and some enzyme patterns in phorbol ester-induced chronic lymphocytic leukemia cells


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

Leukemic cells from 10 patients with B-chronic lymphocytic leukemia (B-CLL) were isolated and cultured in the presence of 12-0-tetradecanoylphorbol 13-acetate (TPA) at a concentration of 8 x 10[-7] mol for 72 hours. Cells were analyzed before cultivation and after 72 h of cultivation with and without TPA for changes in surface membrane (Sm) and cytoplasmic (cyt) markers expression, presence of receptor for mouse rosette forming cells (MRFC) and some enzyme profiles. All B-CLL cases studied showed typical B-cell phenotype. TPA treatment induced hairy cell leukemia (HCL) characteristics, given by the membrane CD22 and CD25 expression and TRAP positivity in the majority of the cases tested. Cells had hairy cell-like morphology with more intensive cytoplasmic immunoglobulin (CIg) fluorescence staining, absent receptor for MRFC and increased activity of purine nucleosidephosphorylase. In common these changes indicate that TPA can induce hairy cell characteristics on B-CLL cells in vitro suggesting the more mature differentiation stage of HCL compared with CLL. Furthermore, we originally demonstrated that the CD22, present in the cell membrane after TPA, could be detected in the majority of unaffected B-CLL cells in their cytoplasm. From the technical point of view some intracellular CD markers and Igs of B-CLL cells in viable cells in suspension assayed by flow cytometry are described in this study.

Key words: Chronic lymphocytic leukemia, hairy cell leukemia, TPA, surface membrane and cytoplasmic markers.
pp. 221-226

Human hematopoietic cell lines: A model system for study of minimal residual disease detection technique in acute leukemia


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

Double immunofluorescence studies using both surface and cytoplasmic antigens were performed on cells of some human hematopoietic lines. We tested several permeabilization protocols in order to optimize, improve and simplify flow cytometric assay to detect the combinations of two markers present in one cell which could be regarded as leukemia-related markers. It was found, that buffered formaldehyde-acetone (BFA) fixation renders the cell membrane permeable without destroying surface antigens so that intracellular and cell surface markers could be measured simultaneously by flow cytometry. Cell lines used for the experiments reported here included MOLT4 T cell line, mature B cell lines DAUDI and U-266, and early B cell line REH-6. Results from our studies demonstrated, that in the absence of CD3 antigen on the surface membrane of viable MOLT4 blast cells, double labeling of fixed, permeabilized cells revealed 97% mCD7+, cCD3+ double positive cells. Two color staining with anti-CD19 and anti-CD22 monoclonal antibodies (MoAbs) in DAUDI cells showed, that larger part of cCD22+ cells expressed mCD19 antigen. CD22 antigen was absent on DAUDI cell membrane. Of great interest was the finding, that the marker detected by anti-CD19 MoAb which was absent on the membrane of U-266 cells was detected in their cytoplasm. Double staining of these cells revealed, that the number of mCD22+, cCD19+ double positive cells was 80%. Cytoplasmic CD22 antigen along with surface membrane CD19 was used to define early B cell line REH-6 as well. Our results demonstrate majority of double positive cells among tested population (mCD19+, cCD22+). To our knowledge the presence of cytoplasmic IgM detectable by flow cytometry in REH-6 cells, which could be so regarded as a precise and adequate counterpart to pre-B acute leukemia cell phenotype in children, is an original finding.
Immunological typing plays an important part in the multiple marker analysis of hematopoietic malignancies. Through these surface and cytoplasmic marker combinations minor neoplastic cell populations could be detected. Human hematopoietic cell lines could serve as a reliable model system for monitoring minimal residual disease in acute leukemia patients.

Key words: Human leukemia-lymphoma cell lines, cell fixation and permeabilization, double color flow cytometry, leukemia-related marker combinations.
pp. 227-234

p53 expression in breast cancer related to prognostic factors


Departments of Immunopathology, Radiotherapy, Patomorphology and Oncological Surgery of Medical University of Gdansk, 80-211 Gdañsk, Poland; Division of Radiotherapy, PCK Hospital, Gdynia, Poland

In this article the results of molecular marker p53 examinations were presented in relation to the following established breast cancer prognostic factors: age, histologic type, histologic grade, lymph node involvement, tumor size as well as estrogen a progesterone receptor status. Twenty one percent of these primary breast cancer specimens exhibited the overexpression of p53 protein. Significant associations were found between p53 overexpression and younger age, high histologic grade and low content of estrogen and progesterone receptors. Identification of p53-positive breast carcinomas potentially represents a clinically useful indicator of breast cancer aggressiveness.

Key words: Breast cancer, p53 protein, tumor suppressor genes, estrogen and progesterone receptors, prognostic factors.
pp. 235-237

DNA index as prognostic factor in breast cancer


Department of Cell Biology, Maria Sklodowska-Curie University, 20 033 Lublin, Poland;
¹Drugs Institute, Warszawa, Poland;
²Oncology Hospital, Lublin, Poland

Enzymatically dissociated cells exhibited growth in most cases while mechanically dissociated cells did not grow in short term cultures and were not suitable for cytometric study. Breast cancer cells obtained by enzymatic dissociation were examined for DNA content and S-phase fraction. The most of the primary and metastatic breast cancer cells were aneuploid. The 74% rate of invasive and 62% of primary breast cancers had aneuploid (> G[0]/G[1]) DNA content. 22% of metastatic breast cancer cells and 37% of primary tumors cells were DNA diploid or near diploid tumor stemlines. The means of DNA diploid and near diploid tumor cells were higher in primary tumors. DNA index was higher in more aggressive and metastatic tumors. S-phase fractions were higher in cells of metastatic tumors than those in primary tumors. Nuclear DNA content and S-phase fraction can be considered a valuable prognostic indicators in breast cancer.

Key words: Breast cancer, DNA content, ploidy, flow cytometric DNA analysis, prognosis, cell viability.
pp. 239-242

Micronucleus assay in three transplantable mouse tumors


Laboratory of Radiation Biology, Centre of Oncology, 31-115 Cracow, Poland

The cytokinesis-block micronucleus (MN) assay was performed in three mouse tumors: two sarcomas (SaL, MCA) and Lewis lung carcinoma (LLC). To determine the optimal culture durations and cytochalasin B (cyt-B) concentrations to yield the highest proportion of binucleate cells (BC) for each tumor, the influence of the cyt-B concentration (1, 2 and 3 microg/ml) and culture duration (24-96) were studied. The amount of BC and the MN frequency was investigated for the different radiation doses (0-4 Gy). Dose response curves were constructed using the optimal culture duration and cyt-B concentration for each tumor. This was 24 h of incubation for MCA and 48 h for SaL and LLC and 2 microg/ml of cyt-B. The tumors examined differ in the mean number of spontaneously (0 Gy) occurring MN in binucleate cells. These were 0.008, 0.022 and 0.044 for MCA, SaL and LLC, respectively. The MN frequency increased with radiation dose. LLC was found to be the most radiosensitive, while MCA proved to be the least radiosensitive tumor. The average number of MN/BC at 2 Gy of irradiation (after subtraction of the value at 0 Gy) ranged from 0.05 to 0.36. The highest mean value - 0.36 was shown in LLC, the middle - 0.20 in SaL, and the lowest - 0.05 in MCA tumor. After higher doses of irradiation numerous BC with two and more MN were found in LLC tumor, while they were not frequently observed in MCA tumor.
We did not observe an increase in the MN frequency with culture duration or proliferation rate of the tumor cells. MCA has the shortest potential doubling time (T[pot]) and had the lowest MN frequency from three examined tumors. The MN assay has promise to be a rapid predictive assay of radiosensitivity.

Key words: Micronucleus assay, mouse tumors, radiosensitivity.
pp. 243-248

Phorbol ester (TPA)-induced differential modulation of cell surface antigens in human pluripotential leukemia (K-562) cell line: Effects of protein kinase inhibitors with broad- and PKC selective inhibitory activity


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

Phorbol ester (TPA)-induced increase in cell surface expression of adhesion structures, i.e. intercellular adhesion molecule-1 (ICAM-1, CD54), beta-2 integrin LFA-1 (CD11a), complement-regulatory cell membrane protein-protectin (CD59) and leukocyte common antigen (CD45) in human erythroid/myeloid leukemia cell line K-562 was inhibited by staurosporine, an inhibitor with broad, non-selective protein kinase inhibitory profile, but not by CGP 41 251, a benzoylated staurosporine derivative with the selective protein kinase C (PKC) inhibitory activity. Neither staurosporine nor CGP 41 251 modulated TPA-induced down-regulation of trasferrin receptor (CD71). These data suggest that phorbol ester-induced cell surface antigen modulations in K-562 cells are predominantly mediated by PKC-independent signalling pathways.

Key words: Cell surface antigen-CD54, CD11a, CD59, CD45, CD71 antigens, phorbolester (TPA), modulation, protein kinase inhibitors, staurosporine, CGP 41 251.
pp. 249-253

In vitro antileukemic activity and chemical transformation of the 5'-chloro-5'-deoxy derivative of cyclocytidine


Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University, 832 32 Bratislava, Slovakia;
¹Department of Experimental Therapy, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia;
²Institute of Chemical Drugs, Faculty of Pharmacy, The University of Veterinary Medicine and Pharmacy, Brno, Czech Republic

Hydrochloride of 5'-chloro-5'-deoxy-cyclocytidine (Cl-cC) is an analogue of cyclocytine hydrochloride (cC), a prodrug of the compound with the strong antileukemic activity arabinosylcytosine (araC). This paper is devoted to the study of its cytotoxic activity in vitro and to the effect of acid and alkaline conditions and temperature on its stability. Cl-cC inhibits not only the growth of L1210 leukemia cells in vitro and the DNA synthesis (IC50 = 0.09 micromol/1) but, at the same time, it has a weak effect on RNA synthesis (IC[50] > 250 micromol/l) and no effect on proteosynthesis. In alkaline conditions Cl-cC is transformed to 5'-chloro-araC and 2',5'-anhydro-araC but is more stable in acid solutions.

Key words: 2',5'-anhydro-araU, 5'-chloro-cyclocytidine, 5'-chloro-arabinosylcytosine, cytotoxic activity, stability, transformation kinetics.
pp. 255-258

Assessment of antimutagenic effects of stobadine dihydrochloride on MNNG-induced mutations in Chinese hamster cells V79


Cancer Research Institute of Slovak Academy of Sciences, Spitalska 21, 812 32 Bratislava, Slovakia;
¹Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovakia;
²Institute of Preventive and Clinical Medicine, Bratislava, Slovakia

Mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and antimutagenic effect of antioxidant stobadine (STB) were investigated by so called HGPRT/V79 system. Cells were treated by STB before, during and after MNNG-treatment. Our results showed that the highest antimutagenic effect of STB was observed if the drug was given as a pretreatment before exposure of cells to MNNG. This effect was not concentration-dependent within the framework of 1.5-9 mmol. All other combinations of MNNG- and STB-treatment led to the weaker but statistically significant decrease of 6-TG[r] mutations. Inhibition of proteosynthesis induced by methylxanthine pentoxifylline in the time of pre-MNNG-treatment removed completely antimutagenic effects of STB.
In addition to mutagenicity assays, cytotoxicity of STB and combined effects of MNNG and STB were studied. Trypan blue exclusion and growth activity of influenced cells showed that the application of STB (1.5 mmol) before or after MNNG (0.5 microg/ml) treatment had a similar toxic effect as MNNG alone. Application of STB during MNNG-treatment or pretreatment of cells with STB followed by combined treatment of cells by STB + MNNG statistically significantly decreased viability of cells. There are probably no relationships between the antimutagenic and the toxic effects of combined influence of STB and MNNG on V79 cells.

Key words: N-methyl-N'-nitro-N-nitrosoguanidine, antioxidant stobadine, HGPRT/V79 system, mutagenic and antimutagenic effect.
pp. 259-264

Increased antioxidant enzyme activities in the colorectal adenoma and carcinoma


Research Institute of Nutrition, 833 37 Bratislava, Slovakia;
¹2nd Medical Clinic, Derer's Hospital, 833 05 Bratislava, Slovakia

Most colon carcinomas are preceded by an adenomatous polyp - adenoma-carcinoma sequence. Active oxygen species (AOS) can play a role in the pathogenesis of this process. Antioxidant enzymes (AE) are the primary defense against the deleterious effect of AOS. Activities of AE in 56 individuals with colorectal adenoma (CA), 29 individuals with colorectal carcinoma (CC) and in 24 control subjects were examined. Biopsy specimens from the non-neoplastic colonic mucosa and from the CA and CC were taken during colonoscopy for histological and enzymological analysis. Activities of following AE were estimated: CuZn-superoxide dismutase (CuZn-SOD), catalase (CAT) and glutathione peroxidase (GPx). It was found that individuals with CA and CC were characterized by: (1) increased activities of CAT and GPx in non-neoplastic mucosa, that persisted in some of the patients even after removal of tumors; (2) increased activities of CuZn-SOD, CAT and PGx in CA and CC tissues. It can be inferred that the accumulation of peroxides in the non-neoplastic colonic mucosa induced higher activities of CAT and GPx. The reasons of high activities of all AE in the tissues of CA and CC and their relation to carcinogenesis are not clear and require further studies.

Key words: Colorectal adenoma and carcinoma, antioxidant enzymes, superoxide dismutase, catalase, glutathione peroxidase.
pp. 265-269

Comparison between serum levels of carcinoembryonic antigen, sialic acid and phosphohexose isomerase in lung cancer


**Biochemistry Division, Department of Cancer Biology, The Gujarat Cancer and Research Institute, Asarwa, Ahmedabad - 380 016, India;
¹Molecular Endocrinology Division, Department of Cancer Biology, The Gujarat Cancer and Research Institute, Asarwa, Ahmedabad, India;
²Department of Medical Oncology, The Gujarat Cancer and Research Institute, Asarwa, Ahmedabad, India;
³Department of Surgical Oncology, The Gujarat Cancer and Research Institute, Asarwa, Ahmedabad, India

The identification and application of quantifiable tumor markers as adjuncts to clinical care is a story of both success and failure. The present study compared serum levels of carcinoembryonic antigen (CEA) with total sialic acid/total protein (TSA/TP) ratio and phosphohexose isomerase (PHI) in 192 untreated lung cancer patients as well as 80 age and sex matched controls (44 non-smokers and 36 smokers). CEA values were significantly raised (p < 0.001) in smokers as compared to the non-smokers; whereas, TSA/TP and PHI values were comparable between the two groups of the controls. All the biomarkers were significantly elevated (p < 0.001) in untreated lung cancer patients as compared to the controls. Receiver operating characteristic curve analysis revealed higher sensitivities of TSA/TP and PHI as compared to CEA at different specificity levels between 60% and 95%. Mean values of CEA, TSA/TP and PHI were higher in non-responders compared to the responders. The results indicate that TSA/TP and PHI are superior tumor markers than CEA for lung cancer patients.

Key words: Tumor markers, lung cancer, sialic acid, carcinoembryonic antigen, phosphohexose isomerase.
pp. 271-274

Cancer, cardiovascular mortality, and diet in Italy and the Czech Republic


Department of Environmental Epidemiology, Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16 132 Genova, Italy;
¹Institute of Chest Diseases, Prague, Czech Republic

A descriptive study aimed at comparing mortality and dietary patterns in Italy and the Czech Republic was conducted in the period 1970-1990. Mortality from all causes, all cancers, selected site specific cancers and cardiovascular diseases were found to be generally higher in the Czech Republic than in Italy. The North-South gradients observed within Italy have diminished in the course of the last twenty years, mostly due to a less contained decrease of the mortality from cardiovascular diseases and to a marked increase in cancer mortality for Southern regions compared to Central and Northern regions. The mediterranean diet with many health promoting, possibly protective components, mostly of vegetable origin, is consumed in most parts of Italy, particularly in the South. In contrast, a Central European diet abounding in animal products and lacking in fresh fruit and vegetables is generally followed in the Czech Republic. These differences in diet may play a role in the origin of the observed differences in mortality patterns. Some factors other than diet, such as smoking habits, alcohol consumption, endogenous factors, and occupation, that are not considered here, are known to be involved in the causation of some types of cancer. The results of this study are compatible with the hypothesis of a relevant role played by dietary and other life-style habits in the etiopathogenesis of neoplastic and cardiovascular diseases.

Key words: Mortality, cancer, cardiovascular diseases, diet, epidemiology.
pp. 275-283

Smoking habit and benign breast disease


Department of Oncology and Radiotherapy, Medical University of Gdansk, 80-211 Gdansk, Poland

The possible association between cigarette smoking and the risk of benign breast disease (BBD) was assessed in a case-control study conducted in Gdansk, Poland, between 1990 and 1994. The study compared 160 women with newly diagnosed BBD admitted to the Gdansk Cancer Outpatients Clinic and 160 controls, women from outpatients clinics at the Medical University of Gdansk. There was no convincing evidence of an association, either positive or negative, between various indicators of smoking habit (smoking status, number of cigarettes smoked per day, duration of smoking) and the risk of BBD. Slightly lower relative risks (RRs) of BBD in ex-smokers of 10 or more cigarettes per day (RR = 0.9; 95% confidence interval, CI: 0.4-2.2), and with duration of smoking >= 20 years (RR = 0.8; 95% CI: 0.1-3.4), were also observed in current smokers (RR = 0.8; 95% CI: 0.4-1.5), and (RR = 0.8; 95% CI: 0.1-3.4), but these findings were not statistically significant.

Key words: Smoking habit, benign breast disease risk.
pp. 285-287