Potential carcinogenic and inhibitory activity of compounds isolated from Lilium candidum L.

This paper deals with determination of potential carcinogenic and inhibitory activity of 10 compounds isolated from the ethanolic extract of Lilium candidum L. perfonned by DC polarography. The series of investigated compounds consisted of two spirostanol saponins, two pyroline derivatives, jatropham and its glucoside, flavonol kaempferol, 2-fenylethyl-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside, 2-phenylethylpalmitate and methylsuccinic acid. Carcinogenic, resp. mutagenic activity data for these compounds, with the exception of kaempferol, are not available in scientific literature. All tested compounds were reduced in N,N-dimethylformamide in one or two well defined steps. They also fonned a reversible complex with alpha-lipoic acid, a compound serving as an indicator of carcinogenic activity. The marginal diffuse current values of these complexes served as a criterion of a very low potential carcinogenicity. The inhibitory activity of isolates were studied in the presence of 12-O-tetradecanoylphorbol-13-acetate, a specific tumor promoter for an epidermal carcinogenesis, and in the presence of a polyaromatic substance 7,12-dimetylbenz(a)anthracene known as a carcinogen. The spirostanol saponins possessed the highest inhibitory activity (> 70%) and jatropham (66%). The inhibitory activity of jatropham glucoside was significantly lower (49%). Practically zero inhibitory activity was measured for the 2-phenylethylpalmitate and methylsuccinic acid.

Antiemetic efficacy of three serotonin antagonists during high-dose chemotherapy and autologous stem cell transplantation in malignant lymphoma.

The aim of this study was to compare antiemetic efficacy of three serotonin antagonists, granisetron, tropisetron and ondansetron, during conditioning for autologous stem cell transplantation (ASCT). Forty-five malignant lymphoma patients (mean age 38 years, M:F 30:15), undergoing the highly emetogenic regimen BEAM prior to ASCT, were randomized to receive IV granisetron (G) 3 mg once a day, IV tropisetron (T) 5 mg once a day, or IV ondansetron (0) 8 mg twice daily, for six days. The treatment groups were comparable with respect to age, sex and previous experience of nausea and/or vomiting. Nausea and/or emesis control failure was defined as a nausea lasting > or = 4 hours and/or > or = 3 episodes of vomiting/24 h, emesis control failure as > or = 3 episodes of vomiting/24 h. Both the period of chemotherapy (6 days) and the whole period of observation (10 days) were evaluated. Nausea and/or emesis control failure occurred in 24% of patients during the period of chemotherapy and in 51% of patients throughout the whole period of observation, while emesis control failed in 2% and 27% of patients, respectively. The efficacy of three serotonin antagonists was comparable during the chemotherapy period (5 patients with nausea and/or emesis control failure in the granisetron group, 2 in the tropisetron group and 4 in the ondansetron group,p = 0.40). When evaluating the whole period of observation, the antiemetic response to G and T was significantly better than to O, nausea and/or emesis control failure having occurred in 7 (47%) patients treated with G, 5 (33%) patients treated with T, and 12 (80%) patients treated with O, p = 0.03. The results concerning emesis control failures were similar, G 4 (27%), T 1 (7%), O 7 (47%), p = 0.04. Headache was the only frequent side effect of serotonin antagonists (30% incidence). All three serotonin antagonists sufficiently controlled nausea and vomiting during high-dose chemotherapy (BEAM) administration in 67-87% of patients. In comparison with ondansetron, both tropisetron and granisetron proved to be more effective after ASCT, when emetogenic factors other than chemotherapy alone participated.

Medullary thyroid carcinoma (MTC)--clinical and molecular aspects on the basis of own experience.

In our clinic 19615 patients were operated over 25 years on for goiter. Malignant thyroid neoplasms were found in 1049 (5.3%) patients including 875 (83.4%) women and 174 (16.6%) men. Sixty two adult patients (42 women and 20 men were operated on for medullary thyroid carcinoma (MTC). Thyroid cancer was diagnosed in this group pre or intraoperatively in 44 (71%) patients and postoperatively, on histologic examination, in 18 (29%) patients. These patients were reoperated. Radical operations (total thyroidectomy with regional lymph node removal) were conducted in 43 (69.3%) patients and palliative ones in 19 (30.7%) patients. After MTC surgery, MEN 2A (MTC and an adrenal tumor) were diagnosed by means of imaging techniques (USG, CT) in 6 (9.7%) patients. All adrenal tumors were unilateral. Five of these patients were operated, and pheochromocytoma was confirmed by histopathologic examination. Two years after the MTC operation, 1 women was lost to follow-up. After a year, she was admitted to hospital for severe hypertension and died of cerebral hemorrhagia. Pheochromocytoma was revealed by autopsy. All patients were treated complementarily after the MTC operation. Different combinations of teleradiotherapy, chemotherapy and substitutive doses of levothyroxine were used. Ten (23.2%) of 43 patients operated radically were reoperated 1-3 years after the first operation due to loco-regional tumor recurrence. Radical reoperations were performed in 4 patients, and palliative ones in 6. Over a 0.5-23-year follow-up period, 26 (41.9%) patients died, including 20 of cancer, and 6 of other reasons. Four out of 36 living patients have clinical or biochemical symptoms of neoplastic disease. The follow-up period of MEN 2 patients operated on ranged from 1 to 6 years. Up to now, no tumor in the second adrenal gland has been diagnosed in any of these patients. Genetic (molecular) tests performed in 31 out of 36 living patients revealed mutations of RET gene in 4 (12.9%).

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

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.

A double germline mutations in the APC and p53 genes.

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.

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

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 myelomonocytic 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.

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

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.

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

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 ofoxidative 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.

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

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.

Radiation-induced DNA damage and repair evaluated with 'comet assay' in human ovarian carcinoma cell lines with different radiosensitivities.

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.

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

The efficiency of Xenopus laevis egg extract to repair T:G and A:C mismatched base pairs in unmethylated, hemimethylated and fullymethylated 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 fully methylated 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 ofeukaryotic 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 10(8) 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.

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

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.

Diazenes as modificators of drug-resistance in tumor cells.

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.

Quality of life in cancer patients treated by chemotherapy.

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.

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

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.

Colorectal carcinoid tumors--own experience.

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 Lódź, 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.

PS2 protein in breast carcinomas: cut-off value of estrogen-regulated expression.

This study includes 152 patients with histologically confirmed breast carcinoma. Steroid hormone receptors (SR), estrogen (ER) and progesteron (PR) receptors, and pS2 protein were assayed on the same cytosolic extract in accordance with the recommendation of EORTC. Our results showed menopausal- and histologic grade-related expression of pS2 protein. Unfavorable carcinoma subgroups, in relation to expression of pS2 protein were defined: postmenopausal carcinomas with histologic grade II, and pre-, as well as postmenopausal carcinomas with histologic grade III. There were overlappings of individual pS2 protein values between favorable and unfavorable carcinoma subgroups in relation to the expression of pS2 protein. Otherwise, no overlapping of pS2 protein values was obtained between ER-positive and ER-negative carcinomas within defined unfavorable menopausal - and histologic grade-related expression of pS2 protein subgroups. The highest pS2 protein level observed in ER-negative unfavorable subgroups (15 ng/mg) was considered as the cut-off value which defined estrogen-regulated expression of pS2 protein.

Influence of quercetin on B16 melanotic melanoma growth in C57BL/6 mice and on activity of some acid hydrolases in melanoma tissue.

Quercetin (QC) (5, 7, 3', 4' -tetra oxyflavonolol) is an ubiquitous flavonoid in many plants. The influence of QC on the growth of B16 melanotic melanoma in C57BL/6 mice and activity of some acid hydrolases in the tumor homogenates were investigated. Two series of experiments were carried out: In the first experimental group mice were inoculated s.c. with 10(6) of tumor cells (TC) suspended in 1 ml of saline. TC were obtained from the current serial passages. In the second series of experimental group mice were inoculated with melanoma cells preincubated 15 min. in different concentrations of QC. Mice of both series were divided into three subgroups. Mice of the first series were treated with QC i.p. every second day in a dose of 0.1 mg, 0.5 mg or 1.0 mg (total dose of 1.0 mg, 5.0 mg or 10.0 mg per mice). Animals of the second series did not obtain any treatment. After the nineteenth day of experiment the mice were killed, tumors excised and weighed. Tumor tissue pieces were homogenized for enzyme activity determination. Fragments of tumor tissue were taken for electron microscopy (EM) investigation. In mice injected i.p. with QC mean tumor weight was significantly higher than in control I. The mean tumor weight in the first experimental group was higher than in control from 170% to 196% and in the second experimental group from 69% to 147%. Enzymes activity was also higher in both experimental groups as compared to controls. Arylsulphatase activity in the first group was higher from 102% to 144% and in the second one - from 97% to 115% than in control I. Acid phosphatase activity was higher from 100% to 155% in the first experimental group and from 56% to 161% in the second one. Cathepsin D activity was greater from 133% to 333% and from 113% to 300%, respectively. EM studies revealed the presence of greater number of Golgi structures and primary lysosomes in experimental groups of tumors (mice treated with QC and mice with melanoma preincubated in QC). These results clearly indicate that QC significantly enhances melanotic melanoma growth and increases acid phosphatase and cathepsin D activity in these tumors. The mechanism of QC action on the melanotic melanoma is not fully understood and remains to be defined.

Seasonal variation in estrogen and progesterone receptor levels in breast cancer--a factor in data interpretation.

It is still widely accepted that evaluation of estrogen and progesterone receptors (ER, PR) in breast cancer tissue is one of the basic predictive indicators of hormonal therapy success. Evaluation of hormonal receptors is usually done once at the time of biopsy or surgery on the breast regardless of the effect of time. In a retrospective evaluation of 1301 medical records of breast cancer patients the authors wanted to identify any time period dependence in quantitative or qualitative hormonal status. Steroid receptors were measured in breast cancer tissues by cytosol-based ligand-binding technique. The results suggest a strong time period dependence in hormonal status. In premenopausal patients simultaneous evaluation of ER/PR revealed an increased frequency of both positive receptors in the autumn months, and a decrease in the spring months. In postmenopausal patients, both positive receptors were found more likely in the summer months and both negative in the late winter months. Our results suggest that one evaluation of hormonal receptor status does not necessarily provide an accurate estimate of the hormonal status in breast cancer patients.

Radiation-induced apoptosis and cell cycle progression in TP53-deficient human leukemia cell line HL-60.

Human promyelocytic leukemia (HL-60) cells were irradiated with 0.5-100 Gy of gamma radiation and studied for 48 h post irradiation to determine the mode of death and progression of cells through the phases of the cell cycle. HL-60 cells are much more sensitive to radiation-induced loss of clonogenicity (D0 = 2.2 Gy) than to induction of apoptosis at 6 h (D0 for nonapoptotic cells = 32.6 Gy). After doses 20-50 Gy, the onset of massive apoptosis occurred and nonapoptotic cells were in G1/G0 phase of the cell cycle. In contrast, 6 h after irradiation with doses 2.5-10 Gy maximum cells were in S-phase and 16-24 h after irradiation were arrested in G2-phase. Maximum apoptosis occurred 48 h after irradiation with doses 3.5-10 Gy, and cells that died by necrosis were found in 9-44%.

Intensive cyclic chemotherapy with unprocessed whole blood support in advanced breast cancer.

The aim of our project was to compare the efficacy of mobilised whole blood versus cryopreserved PBPC (peripheral blood progenitor cells) obtained by leukapheresis in the support of hematopoietic recovery in cyclic intensive chemotherapy. Twenty-nine women with breast carcinoma were treated. The mean age was 46 years. In stage III were 23, in stage IV were 6. They received 6 cycles of epirubicin 150 mg/m2 and cyclophosphamide 1250 mg/m2. In the first cycle, 24 hours after chemotherapy, application of G-CSF 5 microg/kg/day was started, and discontinued when leukaphereses and whole blood collections were done. Leukapheresed progenitors were then divided into 3 aliquots, cryopreserved and reinfused after the 4th, 5th and 6th chemotherapy cycles. Mobilised whole blood was collected on day 14 of the 1st and 2nd cycles and reinfused 24 hours after chemotherapy. The occurrence of grade IV leukopenia was 1.82 times higher with whole blood support and grade IV thrombocytopenia 2.64 times higher than in cycles with cryopreserved PBPC support. This resulted from the fact that in one application the numbers of CD34+ cells and CFU-GM were nearly double in cryoconcentrates. The yields of CD34+ cells in 450 ml of whole blood were 1.8 x 10(6)/kg, which is not sufficient for optimal hemopoietic recovery.

Peripheral progenitor cells (PBPC) in supportive care after high-dose chemotherapy in breast cancer.

Hemopoietic growth factors (HGF) and leukapheresed peripheral progenitor cells (PBPC) are increasingly used for supportive care in high-dose chemotherapy (HDC) of solid tumors. Presently, therapeutic protocols with cyclic HDC plus PBPC support are successfuly used in breast cancer patients. Administration of PBPC significantly influences hemopoietic recovery in terms of shortening the pancytopenia period which reduces the risk of dangerous complications, especially the risk of infection. As a certain controversy exists about efficacy of this therapy, large randomized studies are conducted to find more accurate conclusions. In 1998 National Cancer Institute (NCI) gave top priority to four randomized studies of HDC with PBPC support. In recent years, rising yields of PBPC are obtained. The use of new combinations and dosages of hemopoietic growth factors leads to a significant increase of progenitor cells circulating in peripheral blood. Effective mobilization regimens combinations of chemotherapy and cytokines - enable to increase the numbers of circulating progenitors as much as 100-fold. Another aspect, how to minimize the risks is to reduce the transplant volume and so reduce the amount of cryoprotective agent DMSO (dimethyl sulfoxide) and hemolysed erythrocytes. This led to the idea to use only whole blood enriched for PBPC. At present it has been used also in our patients. The results show that enriched whole blood can be used as sufficient substitution for support in intensive cyclic chemotherapy in breast cancer patients.

Estimation of changes in tubulin induced with etoposide in human leukemia cells line K-562 by immunofluorescence microscope.

This study was undertaken to examine the influence of etoposide on the distribution of tubulin in cells of human leukemia cell line K-562 by using immunofluorescence method. The cells were cultured with 5 different doses of etoposide: 0.2, 2, 20, 200, 2000 microM/ml. Changes in tubulin were dependant on concentration of the drug compared to untreated control cells. The changes occurred in distribution of tubulin in cells K-562 after treating them with 200 and 2000 microM/ml etoposide. Cells treated with 200 microM/ml etoposide showed intense diffuse staining of the entire cells. At the surface of the cells there were also intensively stained structures extended outwards from the surface as lamellipodias. Cells cultured with 2000 microM/ml etoposide were much bigger from other cells exposed to lower doses of etoposide and control cells. Distribution of tubulin throughout these cells with diffuse intensive labeling of the cell periphery was seen. Very bright protrusions formed in lamellipodias at the surface of the cells were also observed.

Biological activity of some 4-anilinoquinazolines: cytotoxic, genotoxic and antiprotease effects, induction of necrosis and changes of actin cytoskeleton.

Fourteen substituted 4-anilinoquinazolines have been tested for cytotoxic effect and structure activity relationships. The most active derivatives were substituted by chlorine or bromine group in the aromatic ring, in the pyrimidine ring by morpholine group and in the aniline skeleton by nitro group in position 4 or 2. Derivatives 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline, 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline, 2-(morpholin-1-yl)-4-(4'-bromoanilino)-quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline inhibited growth of tumor cell lines HeLa, B16 and L1210. Mutagenic data provided by Ames test showed, that the compounds 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline and 2-(morpholin-1-yl)- 4-(4'-bromoanilino)quinazoline did not exhibit the mutagenic effect, whereas the compounds 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino) quinazoline increased slightly the number of revertants of the strain TA 98 without metabolic activation. Concentration 26 micromol/L of 6-bromo-2-(morpholin-1-yl)-4-anilinoquinazoline induced necrosis of tumor cells B16. Concentration 5.2 micromol/l induced a significant increase of filamentous actin in the transformed HepG2 cells. Derivatives 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline, 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline, 2-(morpholin-1-yl)-4-(4'-bromoanilino)quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline exhibited antiprotease effect on plasmine. This results could be relevant for the anticancer properties of these compounds.

Signal-averaged electrocardiography in survivors of Hodgkin's disease treated with and without dexrazoxane.

Doxorubicin is one of the most effective anticancer drug, but its usefulness is limited by the risk of developing cardiomyopathy, cardiac dysfunction and ventricular arrhythmias. Dexrazoxane is used to protect against doxorubicin cardiotoxicity. It is uncertain whether the dexrazoxane-mediated cardioprotective effect will be reflected in electrophysiological properties of the heart. The aim of the present study was to evaluate the occurrence of frequency-domain signal-averaged electrocardiographic (SAECG) abnormalities of the QRS complex and the initial ST segment in patients treated with and without dexrazoxane. Thirty children and young adults 2 months - 15 years after completion of doxorubicin-containing therapy for Hodgkin's disease were evaluated with SAECG. Patients from group I (n = 13) received combined therapy with doxorubicin and dexrazoxane (DOX/DZX), patients from group II (n = 17) received doxorubicin without dexrazoxane (DOX). Using fast Fourier transformation within the QRS complex and the initial ST segment, area ratio (AR) values 40-100/0-40 Hz were calculated. Significant differences in these frequency parameters in the QRS complex between DOX/DZX group and DOX group (19.45+/-12.72 vs 46.18+/-43.06; p = 0.03) might indicate protective effect of dexrazoxane on electrophysiological myocardial properties.

Cathepsin B and L and stefin A and B levels as serum tumor markers in squamous cell carcinoma of the head and neck.

Cysteine proteinases cathepsin (Cath) B and L and their endogenous inhibitors stefin (Stef) A and B concentrations were measured using a quantitative immunosorbent assay (ELISA; KRKA d.d., Novo mesto, Slovenia) in serum samples from 35 patients with primary and 7 patients with recurrent squamous cell carcinoma of the head and neck (SCCHN), obtained at diagnosis (Serum no.1) and after therapy (Serum no. 2), and compared to sera from 30 (Stef B, 90) healthy volunteers. A significantly higher Stef A (P = 0.005) and lower Stef B (P < 0.001) concentrations were measured in patients' Serum no.1 than in controls, and the levels of Caths B and L and Stef A were found to be significantly elevated in Serum no.1 as compared to Serum no. 2 (P = 0.045, P = 0.041 and P = 0.024, respectively). The time of Serum no.2 collection did not influence the concentration of either Caths or Stefs in these samples, and no correlation was observed with the established prognostic factors for any of the parameters studied. Patients with subsequently diagnosed recurrent disease had a significantly lower Cath L concentration than those without evidence of relapse during follow up (P = 0.05). The risk of disease recurrence and SCCHN-related death correlated significantly with low Cath L serum levels (P = 0.012, P = 0.006). The serum levels of Cath B, Stef A and Stef B did not influence significantly the probability of survival.

The role of STIR MRI sequence in the evaluation of the breast following conservative surgery and radiotherapy.

The purpose of the study was to define the value of fat suppressed STIR sequence in the MRI of the conserved breast. To our knowledge, this study is the first clinical evaluation of STIR sequence in post-therapy conditions. Forty patients with early (T1-2, N0-1) invasive breast cancer underwent conservative surgery and postoperative radiotherapy. Routine follow-up examinations, including physical examination and mammography were supplemented with breast MRI in all cases 6-166 months (mean 27.6) after initial treatments. Three patients had bilateral cancer. Including follow-up (9 patients) MRI examinations, altogether 53 MRIs were available for analysis. An 0.5 T MRI (Elscint, Haifa, Israel) was used with double breast coil. Axial T1 and T2 weighted spin echo, STIR and 3D gradient echo dynamic sequences were performed. Pre- and postcontrast slices underwent serial subtraction. Twenty-eight circumscribed lesions were identified. All were well visualised on STIR sequence, regardless of histologic nature of lesions. One low grade DCIS was not detected by any sequence. Differential diagnosis between benign and malignant lesions was not possible by STIR sequence alone. STIR sequence was found to be more sensitive in the detection of treatment related breast edema and fluid collection, than T2 SE (spin-echo) sequence. Even the patients who were not good candidates for subtracted contrast enhanced dynamic studies - because of motion artefacts - could have been examined with satisfactory results. STIR is a very sensitive sequence for depicting circumscribed lesions and post-therapy complications, but not suitable for differentiation. It is a useful tool in the follow-up of patients with conserved breast subjected to radiotherapy.

The degree of bone marrow infiltration in patients with hairy cell leukemia treated with splenectomy compatible with long-term hematological remission.

To determine the degree of bone marrow infiltration with hairy cells which is compatible with long-term hematological remission in patients treated with splenectomy, we have investigated 7 patients surviving in hematological remission 61 to 255 months (median 184 months) after splenectomy. As hematological remission has been considered absence of hairy cells (HCs) in the peripheral blood, normalization of peripheral blood cell counts (hemoglobin 120 g/l, white cell count 4.0 x 10(9)/l, absolute granulocyte count 1.5 x 10(9)/l, platelet count 100 x 10(9)/l) and absence of lymfadenopathy and any other activity of the disease. For detection of HCs a very sensitive method of immunostaining with monoclonal antibody DBA.44 in our own modification has been used. Low values of sIL-2R which is considered to be a non invasive marker of tumor burden and activity in HCL were in agreement with the opinion that the bone marrow was the only locality of tumor involvement in splenectomized patients. Infiltration up to 20% with HCs (range 4% to 20%, median 10%) was found to be compatible with long-term hematological remission and long-term overall survival. Patient (No 1) with 30% infiltration of bone marrow with HCs, still normal peripheral blood cell counts, but a very high level of sIL-2R represents extraordinary finding which has been discussed in details.

The importance of interstitial radiotherapy in the treatment of the base of tongue tumors: a retrospective analysis.

The authors have reviewed their experience with interstitial brachytherapy for the base of tongue cancer with the purpose of introducing treatment strategy and technique and presenting results. Between January 1993 and May 1999 twenty-one patients with primary squamous cell cancer of the base of tongue (T1-4N0-2) were treated by interstitial radiotherapy (RT). Seventeen patients with advanced stage cancer received brachytherapy (BT) boost after 60-66 Gy teletherapy and 4 patients with early stage (T1-2N0) were managed by sole BT after tumor excision and elective neck node dissection in case of positive or very narrow (< 5 mm) margin. High-dose rate (HDR) after-loading unit (Ir-192 source) was used with rigid needles or flexible plastic tubes. The treatment plan was performed by PLATO 3D BT planning system. The mean dose of boost BT or sole BT was 20 Gy (12-24 Gy) and 27 Gy (24-30 Gy), respectively. All treatments were delivered on consecutive days with a twice daily fractionation schedule, except the rigid needle technique (n = 4), where the dose was 12 Gy with a single fraction. After definitive RT of advanced stage disease, the rate of complete or partial remission was 65% (11/17) and 35% (6/17), respectively. At a mean follow-up time of 32 months the local tumor control for the entire patient population was 62% (13/ 21). Five patients (24%) died of local and/or regional failure and sixteen patients (76%) are alive (6 with local and/or regional disease and 10 without evidence of disease). All of the four sole BT treated patients belong to the latter group. The incidence of grade 2 or grade 3 mucositis was 48% and 52%, respectively. To achieve good local control with adequate doses, avoiding surgical morbidity and associated functional loss and to minimize late radiation sequelae, the combination of percutan and interstitial RT seems to be very advantageous in the treatment of the advanced tumor of the base of tongue. For patients with early stage (T1-2N0) cancer, sole postoperative BT of the tumor bed - by positive or very narrow margins - seems to be a feasible option. However, more patients and longer follow-up is required to define the value of sole BT.

The efficacy of radiotherapy for vertebral hemangiomas.

Vertebral hemangiomas are benign, slowly growing tumors sometimes causing local pain in the spine and/or neurologic disorders. The present paper includes 14 cases of painful vertebral hemangiomas treated by radiotherapy. All patients were irradiated using standard fractionation scheme with a total dose 20-30 Gy. One month after the treatment complete pain relief was noted in 36% of cases, five months later in 67% of cases, but in the remaining cases partial pain relief was noted. No correlation between treatment outcome and different biological and technical factors was found. No dose-response relationship was noted. The results suggest that anti-inflamatory effect of radiation plays the major role in this kind of treatment and that radiotherapy for vertebral hemangiomas is easy, short and highly effective analgetic treatment modality.